The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared polyclonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.

In order to assess the clinical manifestations and electrocardiogram (ECG) characteristics of Chinese long QT syndrome (LQTS) patients and describe the phenotype-genotype correlation, the subjects from 5 congenital LQTS families underwent clinical detailed examination including resting body surface ECG. QT interval and transmural dispersion of repolarization (TDR) were manually measured. Five families were genotyped by linkage analysis (polymerase chain reacting-short tandem repeat, PCR-STR). The phenotype-genotype correlation was analyzed. Four families were LQT2, 1 family was LQT3. Twenty-eight gene carriers were (14 males and 14 females) identified from 5 families. The mean QTc and TDRc were 0.56±0.04 s (range 0.42 to 0.63) and 0.16±0. 04 s (range 0.09 to 0.24) respectively. 35.7% (10/28) had normal to borderline QTc (≦0.460 s). There was significant difference in QTc and TDRc between the patients with symptomatic LQTS and those with asymptomatic LQTS, and there was significant difference in TDRc between the asymptomatic patients and normal people also. A history of cardiac events was present in 50% (14/28), including 9 with syncope, 2 with sudden death (SD) and occurred in the absence of β-blocker. Three SDs occurred prior to the diagnosis of LQTS and had no ECG record. Two out of 5 SDs (40%) occurred as the first symptom. Typical LQT2 T wave pattern were found in 40% (6/15) of all affected members. The appearing-normal T wave was found in one LQT3 family. Low penetrance of QTc and symptoms resulted in diagnostic challenge. ECG patterns and repolarization parameters may be used to predict the genotype in most families. Genetic test is very important for identification of gene carriers.

The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hDGF-5 gene was gained by TR-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of TR-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells.

Whether surfactant protein B (SP-B)-18A/C and 1580C/T polymorphism were associated with susceptibility to chronic obstructive pulmonary disease (COPD) in Chinese Han population was investigated. After genomic DNA was isolated from blood of COPD smokers and control smokers, the genotypes of SP-B-18A/C and SP-B1580C/T polymorphism loci were determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) respectively. The results showed that there was significant difference in genotypes distribution frequency of SP-B1580C/T polymorphism locus between COPD smokers and control smokers. C→T mutation rate (including TT homozygote and CT heterozygote) in COPD smokers was higher than in control smokers (57.9% vs 41.7%,x²,P<0.05), whereas there was no significant difference in genotypes distribution frequency of SP-B1580-18A/C locus between COPD smokers and control smokers. The allele frequency (29.1%) of SP-B1580-18A/C locus is lower than T allel (70.9%) in Chinese Han Population, and the distribution was different from that in Mexican, in which, the A and T allele frequencies were 85% and 15% respectively. It was concluded that SP-B1580 T allele was probably associated with increased susceptibility to COPD in Chinese Han population; The polymorphism of SP-B-18A/C locus maybe varied with race.

In order to investigate whether Arg110Gln polymorphism in the coding region of the IL-13 gene is associated with asthma and total plasma IgE level in Han nationality in Hubei Chinese population, the allele frequency of 4257 (g/a) site and Arg110Gln genotype of IL-13 was detected by using restriction fragment length polymorphism in Han nationality in Hubei Chinese population including 43 asthmatic children, 45 asthmatic adults, 31 control children and 46 control adults. Total plasma IgE was measured by Chemiluminescence assay. The results showed that the frequency of allele A at 4257 bp of IL-13 in children and adults was 0.39 and 0.32, respectively. The GlnGln form of Arg110Gln polymorphism of IL-13 gene was associated with susceptibility of asthma and elevated total plasma IgE in children (P=0.030 and 0.0009, respectively), but not with them in adults (P=0.219 and 0.174, respectively). Our results suggest that the Arg110Gln polymorphism of IL-13 gene is associated with susceptibility of asthma and elevated total plasma IgE in Chinese children of Han nationality in Hubei, but not with them in adults.

The pathogenesis of hyperoxia lung injury and the mechanism of amygdalin on type 2 alveolar epithelial cells (AEC2) isolated from premature rat lungsin vitro were investigated. AEC2 were obtained by primary culture from 20-days fetal rat lung and hyperoxia-exposed cell model was established. Cell proliferating viability was examined by MTT assay after treatment of amygdalin at various concentrations. DNA content and the proliferating cell nuclear antigen (PCNA) protein expression of AEC2 were measured by using flow cytometry and immunocytochemistry respectively after 24h of hyperoxia exposure or amygdalin treatment. The results showed that hyperoxia inhibited the proliferation and decreased PCNA protein expression in AEC2 of premature ratin vitro. Amygdalin at the concentration range of 50–200 μmol/L stimulated the proliferation of AEC2 in a dose-dependent manner, however, 400 μmol/L amygdalin inhibited the proliferation of AEC2. Amygdalin at the concentration of 200 μmol/L played its best role in facilitating proliferation of AEC2sin vitro and could partially ameliorated the changes of proliferation in hyperoxia exposed AEC2 of premature rat. It has been suggested that hyperoxia inhibited the proliferation of AEC2s of premature rat, which may contribute to hyperoxia lung injury. Amygdalin may play partial protective role in hyperoxia-induced lung injury.

The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats were randomly assigned to two groups: control group and RA group. The rats in RA group was intraperitoneally injected with all trans-retinoic acid (500 μg/kg every day) for consecutive 3 days after birth, while those in the control group were not subjected to intervention. Immunohistochemical assay was performed to locate the expression of PDGF. mRNA levels of PDGF were measured by reverse transcription polymerase chain reaction (RT-PCR) at age of 1, 3, 5, 7, 10, 14, 21 days. The method of radial alveolar counts (RAC) was used to measure the amount of the alveoli of the lungs. It was found that with increasing days, levels of PDGF-A and PDGF-B changed to verying degrees. RA could elevate significantly the expression levels of PDGF-A mRNA and protein (P<0.01), but not affect the expression levels of PDGF-B mRNA and protein markedly (P>0.05). It is suggested that PDGF might play an important role in lung development. RA can stimulate lung development through increasing the expression levels of PDGF-A mRNA and protein.

The inhibitive effects of all-trans retinoic acid (ARTA) on airway inflammation in asthmatic rats and its mechanism on the basis of the regulation of nuclear factor kappaB (NF-κB) were explored. Thirty-two SD rats were randomly divided into 4 groups: control group, asthma group, dexamethasone treatment group and retinotic acid treatment group. The total and differential cell counts in the collected bronchoalveolar lavage fluid (BALF) were measured. The pathological changes in lung tissues were estimated by scoring. The expression of NF-κB inhibitor (IκBa), NF-κB, intercellular adhering molecule 1 (ICAM-1) in lung tissue was detected by immunohistochemical method. The results showed that in the two treatment groups, the total cell counts and proportion of inflammatory cells in BALF were significantly reduced, but there was no significant difference in differential cell counts in BALF between them. The pathological changes in lung tissues in the treatment groups were significantly attenuated as compared with asthma group. Except the epithelial injury in retinotic acid treatment group was milder than in dexamethasone treatment group, the remaining lesions showed no significant difference between them. In the two treatment groups, the expression of IκBa was increased, while the expression of NF-κB and ICAM-1 decreased with the difference between the two groups being not significant. It was concluded that the similar anti-inflammatory effects and mechanism of ATRA on airway in asthmatic rats to those of dexamethasone were contributed to the increase of cytoplasmic IκBa content and suppression of NF-κB activation and expression.

The preventive mechanism of salviae miltiorrhizae (SM) against experimental atherosclerosis (AS) in rabbits models was investigated. The experimental AS rabbit models were reproduced by feeding the high cholesterol diet. The changes of atherosclerotic plaques in normal group, model group and SM treated group were observed. The levels of serum TG, TC, HDL-C and LDL-C were determined. The immunohistochemistry was used to detect the expression of Bcl-2, Bax and IL-6 proteins in atherosclerotic plaques. The results showed that the level of serum TG in SM treated group was significantly lower than in model group (P<0.01). Immunohistochemistry revealed that the expression of Bcl-2, Bax and IL-6 in model group was significantly higher than in normal group. In the SM group, the expression of Bcl-2 protein was up-regulated and that of Bax was down-regulated. It was suggested that SM could inhibit formation of AS in experimental rabbits. To decrease the expression of Bax and increase the expression of Bcl-2 protein may be one of the mechanisms of SM against atherosclerosis.

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34⁺ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.

The effects of ligustrazine on the expression of LFA-1, ICAM-1 in bone marrow tissue and the mechanism promoting hematopoietic reconstitution following bone marrow transplantation (BMT) were investigated. The 150 mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. The normal group received no treatment, while in the saline group and ligustrazine group, the mice were subjected to normal saline (0.2 ml, twice a day) and ligustrazine (0.2 ml, twice a day) respectively through a gastric tube. At the 7th, 14th, 21st and 28th day after BMT, survival rate, colony forming unit of spleen (CFU-S), peripheral blood cells and bone marrow mononuclear cells (BMMNC) were measured, histological changes in bone marrow tissue were observed and the expression level of LFA-1, ICAM-1 was detected. In ligustrazine group CFU-S counts on the 10th day and the peripheral blood WBC, PLT. BMMNC counts, hematopoietic tissue volume as well as the expression level of LFA-1 on the 7th, 14th, 21st, 28th day after BMT were higher than in saline group (P<0.01or P<0.05). Mature RBC volume and the expression level of ICAM-1 were significantly lower in the ligustrazine group than in the saline group (P<0.01orP<0.05). In the ligustrazine group, fat tissue volume was higher on the 7th, 14th day after BMT (P<0.01) and was lower on the 21st, 28th day (P<0.01) after BMT than in the saline group. It was concluded that Ligustrazine could improve bone marrow microenvironment and promote hematopoietic reconstitution.

The relationship between glutathione S-transferases (GSTs) M1, T1 genotype and childhood acute lymphoblastic leukemia (ALL) was investigated. GSTM1 and GSTT1 genotypes in genomic DNA from 67 children with ALL and 146 healthy controls were analyzed by using the multiplex polymerase chain reaction (PCR). The frequencies of GSTM1, M1-T1 null genotypes in ALL children were significantly higher than in the healthy controls (76. 12% versus 52.74%. OR=2. 856.P<0.001: 50.74% versus 24.66%, OR=3.148,P<0.01, respectively). However, there was no significant relationship between GSTT1 null genotype and ALL of children (61.19% versus 49.32%. OR=1.621.P>0.05). It was suggested that GSTM1 null genotype might be a risk genotype of childhood ALL. While there as no correlation between GSTT1 null genotype and childhood ALL.

The expression of the constimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28', CD4' CD28 in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80⁺, CD19⁺ CD80⁺ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.

The role of CD80/CD86 and CTLA-4 in the pathogenesis of systemic lupus erythematosus and their clinical significance was investigated. By using RT-PCR technique, the expression of CD80/CD86 and CTLA-4 mRNA in peripheral blood mononuclear cells (PBMC) were semiquantitatively detected in 32 patients with active SLE. The results showed that the percentage of positive CD86 expression in active SLE was increased significantly as compared with normal controls (90.63% vs 60.00%,P<0.01). The mean level of CD86 mRNA expression in active SLE group was markedly higher than in the normal controls (0.6410+0.0174 vs 0.4510+0.0402,P<0.001). Compared with normal controls, the percentage of positive CTLA-4 expression and the mean level of CTLA-4 mRNA expression in active SLE group were both increased significantly (bothP<0.01). There was no statistical differences in positive rate of CD80 and the average level of CD80 mRNA between the two groups (bothP>0.05). It was concluded that the aberrant expression of CD86 and CTLA-4 might play an important role in the activity and development of SLE.

The role of HO-1 inducer, hemin, in chronic renal failure (CRF) rats and its possible mechanism of action was studied. 5/6 subtotal nephrectomy was performed to establish chronic renal failure model. Rats were randomly assigned to 4 groups: sham-operated group, CRF group, ferrous gluconate group and hemin group. At the 10th week after operation, serum creatinine, BUN, RBC, HGB and HCT were measured. Renal pathologic changes were observed. RT-PCR and immunohistochemistry were used to detect the expression and distribution of HO-1. RT-PCR and radioimmunoassay was used to determine the expression of ET-1 in the kidney and plasma. The results showed that as compared with CRF group, serum creatinine and BUN in hemin group were reduced significantly and nephrogenic anemia was improved markedly. Glomerular mesangial proliferation and interstitial lesion were also ameliorated significantly. Hemin not only increased the expression of HO-1 but also reduced the expression of ET-1 in the kidney. The level of ET-1 protein in the plasma was also reduced after hemin treatment. Most of these indexes were not obviously changed in ferrous gluconate group. It was suggested that through inducing the expression of HO-1 and reducing the level of ET-1 in the kidney and plasma, hemin plays an important protective role in 5/6 subtotal nephrectomized rats.

The effects of the combined use of angiotensin converting enzyme inhibitor (ACEI) benazepril and angiotensin II type 1 receptor antagonist (AT1RA) valsartan on apoptosis and the expression of apoptosis-related proteins Fas and FasL in the kidney of rats with adriamycin-induced nephritic glomerulosclerosis was investigated. Uninephrectomy and the injection of adriamycin induced the rat model of glomerulosclerosis. Benazepril (6 mg/kg), valsantan (20 mg/kg), or benazepril (3 mg/kg) plus valsantan (20 mg/kg) was respectively delivered daily by gavage to the rats in three treatment groups for 12 weeks. Apoptosis was examined by means of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL). Immunohistochemistry was adopted to detect the expression of Fas and FasL. Software of pathological analysis quantitated the levels of Fas and FasL. The results showed that as compared with those in the control group, the kidneys in the model group had more severe glomerulosclerosis, much more apoptotic cells and higher levels of expression of Fas and FasL. The degree of glomerulosclerosis, the number of apoptotic cells and the levels of expression of Fas and FasL were reduced by benazepril and valsartan. The combined use of benazepril and valsartan had the best therapeutic effect. It was concluded that benazepril and valsartan could suppress the excessive apoptosis of kidney cells by lowering the expression of the apoptosis-related proteins Fas and FasL, so as to postpone the process of glomerulosclerosis. The combined use of benazepril and valsartan has better therapeutic effect.

In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cellsin vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHAin vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cellsin vitro.

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1–12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

In order to explore the PAR-1 mRNA and protein expression around hemotoma following intracerebral hemorrhage and the relation between the PAR-1 expression and thrombin, collagenase VII was stereotaxically injected into right caudate nucleus in rats. The PAR-1 mRNA expression was detected by RT-PCR method and the PAR-1 protein expression by immunohistochemical method respectively. It was found that the PAR-1 mRNA and protein expression around hemotoma was increased at 6 h after intracerebral hemorrhage (P<0.05), peaked at 2 days (P<0.01), and then declined. The change pattern of the PAR-1 mRNA and protein expression was similar to that of intracerebral hemorrhage after thrombin intracerebral injection. The PAR-1 mRNA and protein expression in hirudin group showed no significant, difference with control group. These results indicated that the PAR-1 mRNA and protein expression were markedly increased after intracerebral hemorrhage, which may be closely related to thrombin. Upregulation of the PAR-1 expression may involve in neurotoxic injury induced by thrombin.

The changes of tumor necrosis factor-α (TNF-α) and brain ultrastructure during cardiopulmonary resuscitation and the effects of ulinastation injection were observed, and the mechanism was investigated. Twenty-four adult healthy Sprague-Dawley rats were randomly divided into control group (8 rats), resuscitation group (8 rats) and ulinastatin (UTI) group (8 rats). Rats in control group underwent tracheotomy without clipping the trachea to induce circulatory and respiratory standstill. Rats in resuscitation and ulinastatin group were subjected to the procedure of establishing the model of cardiopulmonary cerebral resuscitation (CPCR). Rats in ulinastatin group were given with UTI 104 U/kg once after CPCR. In the control group, the plasma was collected immediate, 30 min, 2 h, 4 h, and 6 h after tracheotomy. In resuscitation group and UTI group, plasma was collected immediate after tracheotomy, 30 min, 2 h, 4 h and 6 h after successful resuscitation. The plasma levels of TNF-α were determined by radioimmunoassay (RIA). At the end of the experiment, 2 rats were randomly selected from each group and were decapitated. The cortex of the brain was taken out immediately to observe the ultrastructure changes. In control group, there were no significant differences in the level of TNF-α among different time points (P>0.05). In resuscitation group, the level of TNF-α was increased obviously after resuscitation (P<0.01) and reached its peak 2 h later after resuscitation. An increasing trend of TNF-α showed in UTI group. There were no differences in TNF-α among each sample taken after successful resuscitation and that after tracheotomy. The utrastructure of brains showed the injury in UTI group was ameliorated as compared with that in resuscitation group. In early period of CPCR, TNF-α was expressed rapidly and kept increasing. It indicated that TNF-α might take part in the tissue injury after CPCR. The administration of UTI during CACR could depress TNF-α and ameliorate brain injury. By regulating the expression of damaging mediator, UTI might provide a protective effect on the tissue injury after CPCR.

By culturing bone marrow mesenchymal stem cells of rabbits with fibrin gluein vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4–6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-β3. IGF I. Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum. Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when inducedin vitro and can be used as optimal seed cells for cartilage tissue engineering.

The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414. 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

In order to study the roles of HBx and calcium inhibitor verapamil in apoptosis of human normal hepatic cells, L02-off, a pTet-off stably integrated human hepatic cell line was established, in which HBx expression was tightly induced by Doxycycline. The effect of different amounts of HBx and verapamil on apoptosis of human normal hepatic cells was detected. The study showed that apoptosis was triggered by accumulation of intracellular HBx, while verapamil had no effects on the apoptotic process. It was concluded that apoptosis mediated by HBx was dose-dependent but calcium-independent.

The role of nuclear factor kappaB in intestine injury induced by hepatic ischemia reperfusion was investigated. Eighteen male Wistar rats were divided into 3 groups randomly: sham operation group (group A), hepatic ischemia reperfusion group (group B) and hepatic ischemia reperfusion plus pyrrolidine dithiocarbamate (PDTC) group (group C). The rats in group A were only subjected to laparotomy, those in group B underwent partial hepatic ischemia reperfusion (ischemia for 1 h and reperfusion for 2 h) and those in group C underwent the same procedure as that of group B but received PDTC 200 mg/kg i. v. before and after ischemia. After reperfusion, tissues of jejunum and venous blood were obtained for measurement of TNF-α, MDA and MPO. The levels of TNF-α in jejunum and venous blood, the levels of MPO in jejunum in group B were significantly higher than those in group A and group C (P<0.05). There was no significant different in the levels of MDA between group B and group C. The severity of histological intestinal injury in group B and group C was similar. Hepatic ischemia reperfusion caused intestine injury, NF-kappaB may play an important role in this course and the targeting of upstream components of the inflammatory response, such as NF-kappaB, may have important therapeutic applications.

The effects of different concentrations of vascular endothelial cell growth factor (VEGF) on the fibrovascular ingrowth into rabbits hydroxyapatite orbital implant were investigated. Twelve New Zealand white rabbits were divided into 3 groups and received hydroxyapatite orbital implant surgery in their right eyes. Before and after the operation, the implants were treated with 10 ng/ml VEGF, 100 ng/ml VEGF, or normal saline as control group. The animals received technetium bones scan at 2, 4, and 6 weeks postoperatively. The mean radioactivity counts within region of interest (ROI) of the surgery eye (R) and the non-surgery eye (L) in the same animal were tested, and the R/L ratios were calculated. The implants were harvested at 6th weeks and examined histopathologically. The results showed that at second week, there was no significant difference in mean R/L ratios between VEGF group and control group (F=2.83,P=0.111); At 4th week (F=7.728,P=0.011) and 6th week (F=7.831,P=0.011) postoperatively, the mean ratios in VEGF groups were significantly higher than that in control group. At 6th week postoperatively, the fibrovascularization rates in VEGF groups were higher than in control group significantly (F=8.711,P=0.008). It was suggested that VEGF could promote the fibrovascular ingrowth into hydroxyapatite orbital implant, thus might shorten the time required for complete vascularization of the HA orbital implant.

In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.

The inhibitory mechanism of interferon-gamma (IFN-γ) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-γ on the expression of transforming growth factor beta receptor I in thein vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-γ could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship:Y=1937.5-134.2 Igx (r=−0.971,P<0.01). It was concluded that IFN-γ could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-γ may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.

To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-γ), and macrophage inflammatory protein-3 alpha (MIP-3α) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL-17, IFN-γ, and MIP-3α in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1.1416±0.0591, which was significantly higher than that in normal controls (0.8788±0.0344,P<0.001). The expression levels of IFN-γ mRNA were 1.1142±0.0561 and 0.9050±0.263, respectively, with significant difference (P<0.001). And the expression levels of MIP-3α mRNA in psoriatic lesions was 1.1397±0.0521, which was markedly higher than that in normal controls (0.8681±0.0308,P<0.001). These findings indicate that up-regulated expression of IL-17, IFN-γ, and MIP-3α might be involved in the pathogenesis of psoriasis.

In order to explore the possible role of CC chemokine ligand 20 (CCL20) and its receptor CC chemokine receptor 6 (CCR6) in the pathogenesis of psoriasis, the expression levels of mRNA of them in psoriatic lesions were investigated. The skin biopsies were collected from skin lesions in 35 cases of psoriasis vulgaris and 18 normal controls. RT-PCR was used to semi-quantitatively analyze the mRNA expression of CCL20 and CCR6 in the psoriatic lesions and the normal skin tissues. The results showed that the mRNA of CCL20 and CCR6 was present in every specimen. The expression levels of CCL20 mRNA in skin lesions were 1.1397±0.0521, which were greatly higher than those in normal controls (0.8681±0.0308) (P<0.001). The expression levels of CCR6 mRNA in skin lesions were 1.1103±0.0538, significantly higher than in the controls (0.9131±0.0433,P<0.001). These findings indicate that up-regulated expression of CCL20 and CCR6 mRNA might be related to the pathogenesis of psoriasis.

The diagnostic value of 16-slices spiral computed tomography (CT) for portal vein disorders was evaluated. Forty-one patients were scanned by the 16-slices spiral-CT. The celiac trunk, portal vein and their branches were reconstructed by volume rendering (VR), multiplanar volume reconstruction (MPVR) and maximum intensity projection (MIP) technique, and the results were compared with digital subtraction angiography (DSA). VR, MPVR and MIP could display celiac trunk, portal vein, inferior vena cava and their branches and extent of portal vein-vena cava shunt, portal vein emboli and the fistula of hepatic artery-portal vein. The results from 16-slices CT were better than DSA and identical with pathologic ones. The vessel three-dimension reconstruction technique of 16-slices spiral CT is valuable for evaluating the portal systemic disorders.

In order to study the association between lower urinary tract infection and cystitis glandularis (CG), 120 cases of CG were diagnosed by cystoscopic biopsy in the suspicious foci of the bladder. Among them, 72 cases were subjected to bacterial counting culture of urine and microscopic examination of urinary sediment, and 60 cases to fluorescence quantitative polymerase chain reaction (FQ-PCR) assay to detect HPV, CMV and HSV DNA in urine samples. In the 72 cases of CG, the positive rate of bacterial counting culture of urine was 15.3% (11/72), and gray zone rate was 18.1% (13/72). 31.9% (23/72) patients were positive in bacterioscopy of urinary sediment. There was statistically significant difference as compared with the control group (P<0.01). Only 4 of 60 urine samples were positive by FQ-PCR in detection of the three viruses mentioned above with the positive rate being 6.67%. Compared with the control group, there was no significant difference (P>0.05). It was concluded that the genesis of CG was closely correlated with the chronic lower urinary tract infection, especially caused by Esch coli.

The effectiveness and safety of ureteroscopic holmium: YAG laser lithotripsy for managing ureteral calculi was evaluated. Ureteroscopic holmium: YAG laser lithotripsy was performed in 168 ureteral calculi (upper 27 cases, middle 33 cases and lower 108 cases). The results showed that the stone-free rate was 92.6% in the upper ureteral calculi, 93.9% in the middle ureteral calculi and 94.4% in the lower ureteral calculi, respectively. The complication rate was 4.8% (8 cases). It was suggested that ureteroscopic holmium: YAG laser lithotripsy is a highly effective and safe treatment modality for managing ureteral calculi.

In order to study the character of periodontal ligament cells (PDLCs) attaching on commercially pure titanium (cpTi) by morphology and metrology on the early stage (24 h), 1×10⁵/ml PDLCs in 2 ml culture medium were seeded on cpTi discs fixed in 24-well culture plates. Morphology of cell attachment was observed by contrast phase microscope, scanning electron microscope (SEM) and fluoroscence microscopy. Cell adhesion was analyzed by MTT at 0.5, 1, 2, 4 h respectively. PDLCs could attach and spread on cpTi discs. SEM showed that PDLCs had pseudopod-like protuberance. PDLCs showed different attaching phases and reached saturation in cell number at 2 h. It was concluded that PDLCs had good biocompatibility with cpTi, and showed a regular and dynamic pattern in the process of attaching to cpTi.

In the experimental study on the electrophysiology and functions of heart in the animal, the thorax should be opened to expose the heart, at the same time natural breathing is maintained instead of artificial breathing. The key procedure of this new method is to avoid injuring the pleural cavity, so pneumothorax can be prevented. By means of this new method some subject studies have been finished.