The model of SD rats ligated at the proximate left anterior descend (LAD) of coronary was used. The number and dissociation constant of β receptor were studied by using receptor autoradiography to observe the changes in β receptor and the effects of Radix Ginseng Rubra on cAMP in experimental ischemic my-ocardium. The result showed that the number of binding site in simple ligation group (Bmax = 0. 279) was obviously higher than that in sham operation group (Bmax = 0. 093) and the dissociation constant of simple ligation group (Kd = 12. 431) was higher than that of sham operation group (kd = l. 319). There was a significant difference between the two groups (P<0. 05). It proved that the number of β receptor was increased and the activity was elevated in myocardial cell membranes after ligation of LAD. The myocardial cAMP level in simple ligation group [(1293. 96±519. 36) × 10-3 nmol/g] was much higher than that in sham operation group [(774. 44 ±210. 55) × 10-3nmol/g]; but the level of cAMP in ligation group after receiving Radix Ginseng Rubra treatment (805. 02 ±362. 48 pm/g) was obviously lower than that in simple ligation group (P<0. 01), which was close to the result of sham operation. The results indicated that Radix Ginseng Rubra could decrease the cAMP level in ischemic myocardium.
The expression of foreign gene,Schistosoma Japonicum 26 ku antigen (Sj26GST),in Bacillus Calmette-Guerin (BCG),Mycobacterium CM. smegmatis) andEscherichia coli (E. coli) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST inE. coli as template. The Sj26GST cDNA was cloned into the downstream of humanM. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together intoE. coli-Mycobacteria shuttle plasmid pBCG-2000 to construct the expression shuttle plasmid pBCG-Sj26. The recombinant BCG andM. smegmatis mc2155, which were electroplated with pBCG-Sj26, could express Sj26GST and the recombinantSchistosoma Japonicum vaccine BCG-Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS-PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15% and 10% of total bacterial protein in BCG andM. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.
Bispecific antibodies (BsAbs) of anti-CD3 X anti-idiotype (Id) to B-cell lymphocytic leukemia (CLL) were prepared by chemical conjugation and direct hybridization technique of hybridoma and hybridoma without screening markers. The specificity of BsAbs from culture supernatants or ascites was assayed by indirect ELISA and indirect immunoflurescence (IF). The results showed that BsAbs could specifically react with homologous serum IgM from patients with B-CLL and cells carrying CD3 marker respectively. Cell combination test and LDH assay demonstrated that BsAb significantly increased the conjugate formation between lymphocyte activated kill (LAK) cells and Daudi cells, and enhanced the cytotoxic activity of LAK cells against Daudi cells.
The green fluorescent protein (GFP) from the jellyfishAequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus-like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N-terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted.In vitro, fluorescent VLPs could bind to CV-1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPVl VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.
To assess the role of follicle-stimulating hormone receptor (FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125I-FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR-CHO cells). An 18-mer phosphorothioate-endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cD-NA was synthesized. An 18-mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR-CHO cells were cultured in 24-well plates (105 cells/ well) in the absence or presence of 1–20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for125I-FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA (P< 0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of125I-FSH by 13.6±0.8%(P<0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5% (P 0.05) inhibition of125I-FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1± 1.3% (P<0.05) reduction in binding within 24 h. Binding had returned to 52.3 ± 2.3% (P< 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane-bound FSHR.
PCR enzyme linked immune sorbent assay (ELISA) was developed for detection of RT-PCR-amplified cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA. The non-radioactive hybridization was performed in a streptavidin-coated microwell with digoxigenin-labeled PCR products and with biotin-labeled capture probe. PCR ELISA was proved to be expedient, simple, sensitive and safe for identification of CK19-, CEA-RT-PCR products. These results were proven by sequencing.
In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low-serum medium system by a non-perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate-early genes were detected byin situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultratructure (glycogen rosettes, bile canalicula, wheel-like mitochondria and well-developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.
The clonal growth of human acute leukemia cell line (K562) and acute myeloid leukemia cells in the serum-free culture (SFC) was studied in order to establish a SFC system which could replace the effects of serum by using semi-solid methylcellulose culture technique. Our results showed that the clonal growth of K562 cells in semi-solid culture was dependent on exogenous serum. The K562 could be grown in SFC supplemented with 4 major replacing substances. The multifactor and multilevel orthogonal experiment demonstrated that the colony formation was statistically influenced by the 4 replacing substances at various concentrations (P<0.01). Among them, bovine serum albumin had greatest effect on clonal growth of K562 cells with the optimal concentration being 15 mg/L, followed by transferring, cholesterol and insulin with their optimal concentrations being of 150 mg/L, 7.8 mg/L and 7.0 mg/L respectively. SFC system was formed with the 4 substances at their optimal concentrations. Colony formation of the blast cells in 10 patients with acute myeloid leukemia was observed in this SFC system. There was a heterogeneity of acute myeloid leukemia cells among the 10 patients in response to the growth substances. In SFC system, there was a linear relationship between the number of the clonal formation and the count of the added cells, indicating the colony growth of the cells. Primary acute leukemia cells maintained in SFC system in 10 cases could completely form clones. The colony formation number in some cases in SFC system was more than that of the serum-containing culture. The SFC system could partially replace the serum for study of the clonal formation of human leukemia cells.
To study sensitivity of drug resistance indexes and resistance manner in acute myeloid leukemia (AMD, MTT drug sensitivity, growth types of CFU-Lin vitro, Bcl-2 antigen and Bcl-2/Bax ratio and intracellular fluorescence intensity of daunorubicin (DNR) were determined. In 62 cases of AML, the positive coincidence rate was 73% with MTT test and the negative coincidence rate was 70 %. In 3 commonly used drugs, if one drug showed sensitivity, the coincidence remission rate reached 71%. In 51 cases of AML, there were 31 patients in the group of complete remission (CR), in which CFU-L of 29 patients showed independent growth. CFU-L of 2 patients showed no growth. However, there were 20 patients in the group of non-remission (NR), in which CFU-L of 14 patients showed independent growth. CFU-L of 6 patients showed non-growth pattern. Statistical analysis showed significant difference (P<0.05). In 32 cases of AML, the expression rate of Bcl-2 was 59.55%±19.56% in drug-sensitive group, and one was 77.36 %±11.91% in drug-resistant group, respectively (P<0.05). At the same time, the ratio of Bcl-2/Bax was 7.50±5.04 in drug-sensitive group and one was 14.32±8.99 in drug-resistant group, respectively (P<0.05). In 15 case of clinically drug-resistant AML, the fluorescence histogram of DNR showed leftshift of main peak (LSMP) in 12 patients. They were diagnosed as classical drug resistance. Meanwhile, 1 patient showed right-shift of main peak (RSMP) in 3 patients. They were diagnosed as re-growth drug resistance. It is concluded that MTT and CFU-L might be used for prediction of drug sensitivity or resistance when patients were on treatment. Bcl-2 and ratio of Bcl-2/Bax might be associated with the prognosis. DNR histogram could be employed for identify the pattern drug resistance. The strength and weakness of these techniques were discussed.
An immunohistochemical study of T lymphocyte subsets on frozen substituted plastic embedding bone marrow sections obtained from 10 patients with myelodysplastic syndrome (MDS) was presented. The results of qualitative and quantitative immunohistochemical analysis are as follows: (1) Labile antigens of T lymphocytes were well preserved, thus allowing analysis of distribution of T lymphocyte subsetsin situ; (2) the average number of T3, T4 and T8 lymphocyte of the diffuse infiltrate was about 2%, 0.4%, 0.5%, respectively, of all nucleated cells in bone marrow, and T4/T8 of T cells were below 1.0 in patients with MDS; (3) there were cases of RAS showing T lymphocyte aggregation in bone marrow, but no patient exhibited progressive refractory anemia with excess of blasts(RAEB) and RAEB in transformation (RAEBT). These findings indicated that the immunological abnormalities are of importance in the evaluation of pathogenesis and prognosis of MDS.
Immunohistochemistry was used to detect tumor necrosis factor (TNF-α) expression in arterial wall of diabetic rats. It was found that endothelial cells were swollen and markedly proliferative in these vessels and accordingly TNF-α showed strong positive immunohistochemical reaction in endothelial cells qr extracellular intimai matrix of such vessels, which might be caused by the expression and release of TNF-α from monocytes and arterial wall cells stimulated by AGEs. These findings suggested that increased TNF-α expression might be associated with vascular damage and remodeling in diabetes.
The effect of astragalus on the endothelin in serum and lung of the rats with acute lung injury was studied. The results demonstrated that the concentration of endothelin in the lung of the rats in therapy group was lower than that of the injured rats (64.36±5.37 ng/L vs 103.32±4.99 ng/L, P<0.001), and level of serum endothelin was also lower than that of the injured rats (85.35 ng/L vs 113.35 ng/L, P<0.01). PaO2, serum SOD, lung coefficient, ratio of lung wet weight/dry weight in two groups were also significantly different (P<0.01) respectively, and the lung pathological injury in the treatment group were less than that of injury group. So it is concluded that astragalus could inhibit the increase of serum and lung endothelin, thereby playing a protective role in the rats with acute lung injury.
To explore the roles of mitochondria tRNAleu(UURgene mutation at nucleotide 3243 and the activity of cytochromec oxidase in pathogenesis of preeclampsia, 57 patients with preeclampsia and 60 normotension pregnancy women were screened for tRNAleu(UUR) nt3243 A→G mutation with the method of polymers chain reaction (PCR) and restriction fragment length polymorphism. Cytochromec oxidase activity was determined by measuring the rate of cyanidesensitive oxidation of reduced cytochromec using luminosity photographer. The results showed that cytochromec oxidase activity was significantly lower in the preeclampsia group (0.30±0.39/min, n= 32) than that in the controls (0.73± 0.54/min,n = 26, P<0.01). The mitochondria DNA mutation at position 3243 was not found in our series. The results suggested that the decreased activity of cytochromec oxidase might impair the energy production, leading to the mitochondria dysfunction and placenta dysfunction in preeclampsia patients. Mitochondria dysfunction may be involved in the pathogenesis of preeclampsia. The mutation of mitochondria DNA may not be the common contributor of preeclampsia in our series.
To investigate the clinical significance of monocyte chemotactic protein-1 (MCP-1) produced by endometriotic tissues, the endometriotic tissues were taken from 15 patients with endometriosis. MCP-1 mRNA and MCP-1 protein were determined by dot blot analysis and enzyme linked immunosorbent assay (ELISA) in endometriotic cells cultured with or without interleukin-1β (IL-lβ, 2 μg/L), tumor necrosis factor-α (TNF-α, 20 g/L). After exposure to IL-1β or TNF-α, the expression of MCP-1 mRNA in the endometriotic cells (8.635 ±0.826, 7.031 ±0.970, respectively) were significantly higher than that in the control group (4.482±0.435, P<0.05); The expression of MCP-1 protein in IL-lβ and TNF-α group was 4.52±0.09 μg/L,2.87±0.27 μg/L, respectively, which were significantly higher than 1.74±0.16 μg/L in control (P<0.01). The results suggested that IL-l\ and TNF-α could up-regulate the expression of MCP-1 in endometriotic cells, which might be related to the development of endometriosis.
To explore the anticancer mechanism and DNA damages of hydroxyapatite ultrofine powder (HAUFP) on lymphocytes of rats, DNA damages in W-256 sarcoma cells and lymphocytes of rats were measured by single cell gel electrophoresis (SCGE). The results showed that HAUFP damaged DNA of W-256 sarcoma cells obviously but only cause slight damage of DNA of lymphocytes in rats. It is suggested that HAUFP selectively damaged DNA of tumor cells with only mild damage of lymphocyte DNA. HAUFP has powerful anticancer effect and little genetic toxicity.
A new targeting anticancer system was prepared by using hydroxyapatite particles (2 mm in diameter) as carrier material and adriamycin as anticancer agent. The adsorption and release properties of the complexes were assayed by fluorometryin vivo andin vitro and the curative effect on W-256 sarcoma of rat was observed. The results showed that one particle of hydroxyapatite could adsorb approximately 0.08 mg adriamycin and they can maintain a steady and slow release of adriamycin from hydroxyapatite for one month. When hydroxyapatiteadriamycin complexes were implanted into the liver of rat, liver adriamycin concentration at the implanted region was obviously higher than that achieved by injection of adriamycin solution. The locally implanted complexes obviously inhibited the growth of subcutaneous implanted tumor of rat, and increased the survival rate of rat with implanted liver tumor.
C57 inbred mice (n=100) were employed to develop animal models of Lewis pulmonary carcinoma. The study on the tumor inhibition was performed by infiltrative injection of 3.5% Cy or chlorhexidinum of two different concentrations around the tumor respectively. The suryival, survival rate, tumor growth rate, and pulmonary metastasis node number were compared. The results showed that the inhibitory effects of 0.1% and 0.5% chlorhexidinum were the same as that of 3.5% Cy, but the toxic and side effects were obviously reduced as compared with 3.5% Cy. The optimal concentration of chlorhexidinum was 0.5%. This provides a new approach for infiltrative injection of the tumor for clinical use.
Fifty-six SD rats were randomly divided into the normal control group and the operation group. The operating group was subdivided into six groups on the basis of killing time (the 12th h, the first day, 3rd day, first week, 2nd week and 4th week) after vagotomy (VG). The pancreatic tissues were taken for HE and Feulgen staining. The DNA content of pancreatic exocrine cells was determined by a domestically-fabricated computer image analyzing system. In the control group, on the first day or in the first week after VG, the pancreatic samples were taken for transmission electron microscopic examination. The DNA content of pancreatic exocrinal cells was decreased from 1 to 3 days after VG. The secretion was found to be in inhibitory state and One week later, it gradually restored. The results indicated that the proliferation and the function of the SD rat’s pancreatic exocrinal cells were prohibited at initial stage after VG, which might be concerned, at least in part, with dominance and nutrition of vagus.
Transcranial Doppler (TCD) was dynamically performed on 25 patients with subarachnoid hemorrhage (SAH) before and after treatment. In the first 4 days of clinical course, 3 cases demonstrated elevated intracranial pressure (ICP), 6 cases showed stenosis of blood vessels, and 16 cases gave normal findings. At 7–14 days of clinical course, 20 cases showed vascular spasm or narrowing, 4 cases imparted normal findings and 1 patient died of elevated ICP. After the third TCD performed on the 20th day of the clinical course, 5 cases showed mean blood flow velocities (Vm) below normal values, indicating poor cerebral perfusion, 9 cases still had Vm above normal levels, 10 cases yielded completely normal findings. In conclusion, TCD conducted at different phases of SAH can not only demonstrate the functional and pathological status of cerebral blood vessels following SAH, but also reflect the outcome of clinical features. It is very helpful in the evaluation of the causes and the prognosis of SAH.
The effects of the novel GH-releasing hexapeptide, Hexarelin, on cultured human pituitary somatotrophinomas were investigated. Hexarelin (0.01 -100 nmol / L) dose-dependently stimulated GH secretion up to 4.6-fold. Maximal effects occurred with 10 nmol / L. These effects were very similar to those observed with GHRP-6. The effects of Hexarelin were reduced by phloretin, an inhibitor of protein kinase C (PKC). The rate of phosphatidylinositol (PI) hydrolysis was markedly increased by Hexarelin in a dose-dependent manner. These results demonstrated that Hexarelin could directly stimulate GH secretion by human pituitary somatotrophs in a PKC-dependent manner, probably via activation of the PI transduction system.
In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30%–40% of human pituitary somatotrophinomas carry somatic mutations of the gene for the α-subunit of the stimulatory GTP-binding protein, G,(G,α). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30%) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp-positive and 4 of 7 gsp-negative tumors. Moreover, phorbol ester, 1, 2-tetradecanoylphorbol-13-acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp-positive somatotrophinomas, whereas this effect was not observed in gsp-negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase-cAMP-protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross-talk mechanism.
The present study examined the temporal responses and the efficacy of192Ir-HDR endovascular irradiation for preventing smooth muscle cell proliferation of rabbit iliac arteries after PTA with a cutting balloon catheter. Endovascular irradiation with 12 Gy was randomly performed on the one side of iliac arterial segment with the unirradiated side serving as a control. Animals were euthanatized 1, 2, 3, 4, 8 and 12 week(s) after angioplasty. Histopathological and immunohistochemical studies were carried out. Histopathology showed repair of the dissection by cellular accumulation and a striking reduction in the amount of neointimal hyperplasia in the irradiated arteries as compared with control vessels. A peak of PCNA-positive ratio was in neointima of the control arterial segments at a week. 2–4 weeks after irradiation, the neointimal PCNA-positive ratio was still significantly increased in the control arterial segments compared with the irradiated arterial segments. After 8 weeks, PCNA-positive ratio was below 1% in both irradiated arterial segments and the control. Our results showed that the192Ir-HDR afterloading irradiation with a dose of 12 Gy can be considered sufficient for inhibiting neointimal hyperplasia in angioplastized rabbit iliac arteries with cutting balloon catheter.
The fresh eye balls from the accidental death adults were placed on low temperature slicer to cut into slices. The fresh sections was subjected to the direct measurement of retinal thickness by using CMIAS multi-function real color pathologic imaging analysis system. The results showed that in the total of 6 eye balls measured, the mean retinal thickness was 310.92±34.14 μm. It was concluded that it was simple and had high confidence for the fresh eye ball low temperature frozen sections to directly measure the retinal thickness, which provided a histological method for the measurement of retinal thickness and the reference of retinal thickness of Chinese adults.
The effect of Sinomenine on IL-8, IL-6, IL-2 and mIL-2R produced by peripheral blood mononuclear cells was investigated by using cell culture, radioimmunoassay and flow cytometry. It was showed that production of IL-8 and mIL-2R was inhibited, but the levels of IL-6 were enhanced by Sinomenine. Our results also demonstrated that Sinomenine did not have any effect on the production of IL-2. The study demonstrated that Sinomenine was able to regulate the production of cytokines. This may be one of the mechanisms by which Sinomenine works on rheumatoid arthritis.