Our previous study showed that transmembrane TNF-α (TM-TNF-α) had broader tumoricidal spectrum than secretory TNF-α (s-TNF-α). This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G₁ phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

To explore the anti-tumor effect of immunotherapy with recombinant protein vaccine based on FGFR-1 of chicken (cFR-1) in a mouse Meth A fibrosarcoma model, tumor volume and survival rate of the mice were observed at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. Auto-antibodies against self-FGFR-1 were detected by Western blotting and ELISA, respectively. The anti-FGFR-1 antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. Eighteen days after inoculation of tumor cells, the tumor volume was significantly smaller in cFR-1-immunized group than in mouse FGFR-1 (mFR-1) immunized group and normal saline (NS) control group (P<0.05), and the survival time was significantly longer in cFR-1-immunized group than in the control groups (P<0.01). MVD was significantly lower in cFR-1-immunized group than in mFR-1-immunized group and NS group (16.8±5.6 vs 64.6±1.8 and 59.6±8.7, P<0.01). Antibodies against self-FGFR-1 were found in mFR-1-immunized group, the major antibody subclasses were IgG1 and IgG2b. Compared with the two control groups, the numbers of APBCs in cFR-1-immunized group were significantly increased (P<0.01) These results demonstrated that the cFR-1-related anti-angiogenesis protein vaccine could induce the production of auto-antibodies against self-FGFR-1, which futher inhibit angiogenesis and growth of solid tumor.

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20–1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines (IL-2 and IL-12) in serum were measured by ELISA and peripheral blood T lymphocyte subsets CD4⁺, CD8⁺ were analyzed by flow cytometry. Our results showed that spleen lymphocytes isolated from the CS-CpG ODN group of mice showed the strongest proliferation (SI=1.551), and the levels of IgG, IL-2 and IL-12 in serum were higher than those of other groups. Compared with the immunization with CpG ODN, the immunization with CS-CpG ODN gene carrier was more efficient in up-regulating the percentage of CD4⁺T cells and the ratio of CD4⁺/CD8⁺ of mice. It was concluded that CS gene carrier of CpG ODN was much more effective in improving immunity of CpG ODN in mice.

To investigate the effect of interleukin-1β (IL-1β) on I_A and I_K currents in cultured murine trigeminal ganglion (TG) neurons, whole-cell patch clamp technique was used to record the I_A and I_K currents before and after 20 ng/mL I_L-1β perfusion. Our results showed that 20 ng/mL IL-1β inhibited I_A currents (18.3 ± 10.7)% (n=6, P<0.05). I_L-1β at 20 ng/mL had no effect on G-V curve of I_A but moved the H-infinity curve V0.5 from −36.6±6.1 mV to −42.4±5.2 mV (n=5, P<0.01). However, 20 ng/mL IL-1β had effect on neither the amplitude nor the G-V curve of I_K. IL-1β was found to selectively inhibit I_A current in TG neurons and the effect may contribute to hyperalgesia under various inflammatory conditions.

To investigate the effect of the anti-endotoxic part of Radix Isatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide (LPS), the sample solution of F₀₂₂ part from Radix Isatidis was intraperitoneally administered to experimental mice, and the lipopoly-saccharide (LPS) were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F₀₂₂ part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F₀₂₂ part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

In order to investigate the expression of CD40 in endothelial cells (ECs) in a variety of injured conditions and the interventional role of Andrographitis Paniculata isolate (API₀₁₃₄), the thoracic aorta ECs of guinea pigs were cultured in vitro until the third passage, incubated in the presence of media containing xanthine oxidase (XO) and xanthine (Xan) which produced oxygen free radical (OFR group); oxidized-LDL (ox-LDL group); XO, Xan and API₀₁₃₄ (OFR+API₀₁₃₄ group); or ox-LDL and API₀₁₃₄ (ox-LDL+API₀₁₃₄ group). The expression of CD40 in ECs was detected by immunofluorescence assay and reverse transcription-PCR (RT-PCR). The results showed as compared with the control group, the expression of CD40 in ECs in OFR group and ox-LDL group was increased (P<0.01), but attenuated significantly in OFR+ API₀₁₃₄ group and ox-LDL+API₀₁₃₄ group (P<0.05). It was suggested that API₀₁₃₄ could protect atherosclerosis by inhibiting the expression of CD40 molecule in injured ECs.

To evaluate the safety and efficacy of tirofiban, a specific inhibitor of the platelet glycoprotein llb/llla receptor, in the treatment of unstable angina and myocardial infarction without persistent ST elevation (acute coronary syndrome, ACS), a total of 200 patients were randomly assigned to a heparin group and a tirofiban+heparin group on double-blind basis and the treatment effects of the two protocols on ACS were compared when the patients of both groups were taking aspirin at the same time. The composite primary end-point events consisted of death, myocardial infarction, or refractory ischemia. Our results showed that the frequency of the composite primary end point events in 30 days was lower in tirofiban+heparin group as compared with that of heparin group (13.9% vs 29.3 %, P=0.010). The rates of the other composite end point events in the tirofiban+heparin group were also lower than those in the heparin group in 4.5 days and in 30 days. Bleeding complication occurred in 7.0% of the patients receiving heparin alone and in 12.7% of the patients receiving tirofiban and heparin in combination (P=0.1717). The study showed that the incidence of ischemic events in patients with ACS receiving tirofiban+heparin was lower when compared with that of patients who received only heparin and aspirin, suggesting that tirofiban might be of special value in the treatment of ACS.

This study was designed to investigate the expression of aminopeptidase N (APN)/CD13 on intraembryonic AGM stromal cells, and the change of its enzymatic activity after irradiation injury. The expression of APN/CD13 on AGM stromal cells was assayed by RT-PCR and immunihistochemistry. After the stromal cells in AGM region were irradiated with 8.0 Gy of ⁶⁰Co γ-rays, APN/CD13 enzymatic activity was measured by spectrophotometer at different time points. The result showed that AGM stromal cells strongly expressed APN/CD13. The enzymatic activity of APN/CD13 decreased temporarily after irradiation injury, then increased to higher level 4 h after irradiation, and it returned to the pre-irradiation level 24 to 48 h after the irradiation. The enzymatic activity of APN/CD13 was temporarily enhanced after irradiation injury, which might be one of the compensatory mechanisms that promote the hematopoietic recovery after irradiation.

In order to investigate the anti-cancer effects of deguelin and on K562 and K562/ADM cells in vitro and the underlying molecular mechanism and compare the cytotoxicity of deguelin on K562, K562/ADM cells and human peripheral blood mononuclear cells (PBMCs). The effects of deguelin on cell proliferation were assessed by MTT assay. Apoptosis were detected by Annexin V/PI double-labeled cytometry. The effects of deguelin on the cell cycle were studied by a propidium iodide method. Our study showed that deguelin inhibited the proliferation of K562 cell and K562/ADM cell in a time- and dose-dependent manner and had minimal effects on normal human peripheral blood mononuclear cells. The ratio of IC₅₀ value of deguelin of 24 h on K562/ADM cells to K562 cells was only 1.27, which was significantly lower than the ratio of IC₅₀ value of ADM (higher than 20). Deguelin could induce apoptosis of K562 cells and K562/ADM cells. K562 cells were arrested at G₂/M phase while K562/ADM cells were arrested at G₀/G₁ phase. Our results suggested that deguelin was a novel anti-leukemia agents with high efficacy and low toxicity and it is also a promising agent for reversing drug resistance.

The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (K_y) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the K_v activities and membrane potential (E_m) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P<0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. K_v activities of HASM cells were significantly inhibited by PMA, and the E_m became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P<0.01). This effect could be blocked by Ro31-8220 (P<0.01). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced K_v activity. In passively sensitized HASM rings, this effect was more notable.

To explore the relationship between the serum vascular endothelial growth factor (VEGF) level and the severity of obstructive sleep apnea hypopnea syndrome (OSAHS), the concentrations of serum VEGF in 40 OSAHS patients and 9 healthy controls were measured by using ELISA method. Meanwhile the correlation between the concentration of VEGF and parameters of polysomnography (PSG) was examined. Our results showed that the concentrations of VEGF were significantly higher in OSAHS patients with severe hypoxia (536.8±334.7 pg/mL) than in those with mild hypoxia (329.2±174.7 pg/mL) and healthy controls (272. 8±211.0 pg/mL) (P<0.05 for both). The concentrations of VEGF were also significantly higher in OSAHS patients with hypertension (484.5±261.4 pg/mL) than in those without hypertension (311.0±158.4 pg/mL) and healthy controls (272. 8±211.0 pg/mL) (P<0.05 for both). There was a positive correlation between the concentration of VEGF and the apnea hypopnea index (AHI) (r=0.34, P<0.05). It is concluded that the concentration of the serum VEGF is positively related to the severity of OSAHS. The elevated serum VEGF level may be involved in the pathogenesis of the complications of obstructive sleep apnea hypopnea syndrome.

The expression of c-met stimulated by high glucose in human renal tubular epithelial cells and the role of HGF/c-met system in diabetic nephropathy were examined. The proximal tubular epithelial cells were cultured in vitro under different conditions. MTT was used for the detection of cellular proliferation and RT-PCR was employed for measurement of c-met mRNA level. Our results showed that under different conditions, there were no significant differences in the proliferation of proximal tubular epithelial cells 12 h and 24 h after the culuture (P>0.05). The proliferation of proximal tubular epithelial cells showed a significant change 96 h after the culture and the cellular proliferation induced by hepatocyte growth factor (HGF) was very active (P<0.05). Moreover, no significant difference in the expression of c-met mRNA was found 12 h after the culture under different conditions (P>0.05), while 24 and 96 h after the culture, a persistent and significantly higher expression of c-met mRNA was found in HGF-induced proliferation. It is concluded that addition of exogenous HGF could inhibit the apoptosis induced by high-level glucose, promote the proliferation of proximal tubular epithelial cells, and induce the expression of c-met. Our study suggests that local up-regulation of HGF/c-met system plays an important role in the repair of renal damage in diabetic nephropathy.

To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT) primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.. .

To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the morphology of L02/pcDNA3.1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/ pcDNA3.1-HBx; (3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P<0.01) and the apoptosis rate of HepG2/pcDNA3.1-HBx was lower than that of HepG2 cells (P<0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P<0.01), and the expression of XIAP in HepG2/pcDNA3.1-HBx was about 4 times that in HepG2 (P<0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of hepatic carcinoma by regulating the expression of XIAP.

In order to explore the effect and mechanisms of tumor necrosis factor-α (TNF-α) on the activity of the acyl coenzyme A: cholesteryl acyltransferase (ACAT), THP-1 monocytes were cultured and induced to differentiate into macrophages with phorbol ester. TNF-α (60 ng/mL) was added at different time points into the macrophage-containing medium and the ACAT enzyme activity was measured by quantifying the incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters. The expression of ACAT-1 protein and mRNA was respectively detected by Western blotting and RT-PCR in THP-1 macrophages 24 h after treatment with TNF-α (60 ng/mL). The results indicated that ACAT activity in THP-1 macrophages treated with TNF-α was increased in a time-dependent manner. The expression levels of ACAT-1 protein and mRNA were significantly increased in THP-1 macrophages after treatment with TNF-α (P<0.05). It was suggested that TNF-α could increase the activity of ACAT in THP-1 macrophages by up-regulating the expression of ACAT-1 gene.

The roles of multi-drug resistance protein 1 (MDR1), multi-drug resistance related protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) in the multi-drug resistance (MDR) of hepatocellular carcinoma (HCC) were studied. By exposing HepG2 cell line to progressively increased concentrations of adriamycin (ADM), HepG2 multi-drug resistant subline (HepG2/ADM) was induced. The MDR index of HepG2/ADM was detected by using MTT. The expressions of the four MDR proteins in the three cell lines (L02, HepG2, HepG2/ADM) were investigated at mRNA and protein levels by real-time RT-PCR and Western blot respectively. Our results showed that when the ADM concentration was under 100 μg/L, HepG2 could easily be induced to be drug-resistant. The IC₅₀ of the HepG2/ADM to ADM was 282 times that of HepG2. The expression of MDR1 and BCRP mRNA in HepG2/ADM cells were 400 and 9 times that of HepG2 cells respectively while there was no difference in the mRNA expressions of MRP1 and LRP. There was no difference between HepG2 and L02 cells in the mRNA expressions of the four genes. At the protein level, the expressions of MDR1, BCRP and LRP but MRP1 in HepG2/ADM were significantly higher than those of HepG2 and L02. Between HepG2 and L02, there was no difference in the expressions of four genes at the protein level. HepG2/ADM is a good model for the study of MDR. The four genes are probably the normally expressed gene in liver. The expressions of MDR1 and BCRP could be up-regulated by anti-cancer agents in vitro. The MDR of HCC was mainly due to the up-regulation of MDR1 and BCRP but MRP1 and LRP. These findings suggest they may serve as targets for the reversal of MDR of HCC.

This study examined effect of a new intervertebral cervical disc prosthesis in relieving the neurological symptoms and signs, improving the patients’ ability to perform daily activities, reducing pain, and maintaining the stability and segmental motion. From December 2003 to October 2004, 12 patients, who had received 14 replacements of cervical artificial discs, were followed-up for 2 to 8 months (with a mean of 5.2 months). Of them 5 had cervical spondylotic myelopathy and 7 had cervical disc herniation. The patients included 7 males and 5 females, with their age ranging from 35 to 62 y and a mean of 50.3 y. Single-level replacements were performed in 10 cases and 2 cases received two-level replacement. Operation time of the single-level surgery averaged 130±50 min and the time of two-level surgery was 165±53 min on average (from skin incision to skin suturing). Neurological or vascular complications during or after surgery was not observed. Japanese Orthopedic Association scores (JOA scores) increased from 8.6 to 15.8 on average. There was no prothesis subsidence or excursion. Replaced segments were stable and the range of motion was partially restored, being 4.68° (3.6°–6.1°) in flexion and extension position and 3.51° (2.5°–4.6°), 3.42° (2.6°–4.3°) in left and right bending position. No obvious loss of physiological curvature was noted. CT or MRI follow-up showed that excursion was less than 1.5 mm) in 2 of 14 levels and between 1.5 mm and 3 mm) in 1 of 14 levels. No ossification in the replaced levels was observed. It is concluded that satisfactory short-term results were achieved in the 12 cases of artificial disc replacements. Different from anterior cervical discectomy and fusion, the replacement could achieve quick functional recovery and did not lead to the movement limitation of cervical vertebrae. At least a 5-years follow-up was needed to assess the long-term effect of the prosthesis on its neighboring segments.

To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were divided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted, and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1st, 4th, 8th, and the 12th week after the implantation. Their bone formation was detected by X-ray examination, and tissue response was histologically observed. Western blotting was used for the detection of the expression of collagen I (Col-I) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engineering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.

By preventing mesenchymal stem cells (MSCs) from adhering to precoated agarose to create a model of MSC suspension in vitro, we investigated anoikis in MSCs and the role of caspase-3 in the anoikis. The cultured MSCs were randomly divided into 3 groups: the anoikis group, caspase-3 inhibitor group and control group. Before experiment, we coated dishes with 1.5 % agarose; in the anoikis group, MSCs were put into the precoated dishes; and in the inhibitor group, caspase-3 inhibitor and MSCs were also put into the precoated dishes; but there were not intervention in the control group. MSCs were collected at 2 h, 6 h, 12 h and 24 h. The alteration of caspase-3 activity was evaluated by caspase-3 fluorometric assay and western blot analysis. The apoptosis rates were detected by flow cytometry. MSCs were round and suspended sufficiently in the anoikis and inhibitor groups. Caspase-3 fluorometric assay showed that there were significant differences in statistics between the anoikis group and the others (P<0.05). Western blot analysis discovered that caspase-3 expression in the anoikis group was more than that in the control and inhibitor groups (P<0.05). Flow cytometry showed that the apoptosis peak appeared in all the three groups, but it increased dramatically in the anoikis group. The apoptosis rates in the inhibitor and control groups were low and stable. And there were significant differences in statistics between the anoikis group and the others (P<0.05). MSCs will undergo anoikis in suspended condition if they are separated from the extracellular matrix. Caspase-3 inhibitors can suppress caspase-3 activity and reduce the apoptosis rate significantly. Caspase-3 plays a vital part in induced MSC anoikis in vitro. MSCs suspension culture system might be set up with argorose and caspase-3 inhibitor.

This study is to investigate the effect of local phVEGF₁₆₅ injection on sciatic nerve regeneration in the rats and to search for a new way in the further treatment of peripheral nerve injuries. Forty-five adult male Wistar rats received a neurotomy to bilateral sciatic nerves, which were subsequently reconnected with 10/0 epineurial nylon sutures. The injured segments was locally injected with normal saline (group A), or 25 μg of phVEGF₁₆₅ (group B) or 50 μg phVEGF₁₆₅ (group C). Nerve conduction and regeneration were evaluated in terms of the histological changes, weight of gastrocnemius muscles, electrophysiology and morphometric results. Our study demonstrated that rats of group C showed the best results in terms of nerve regeneration, followed by group B and group A. Our findings suggested that local injection of phVEGF165 can facilitate nerve regeneration and promote functional recovery in a dose-dependent manner.

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.

To investigate the safety, feasibility and effectiveness of transrectal high-intensity focused ultrasound (HIFU) in the ablation of canine prostate, 20 dogs were divided randomly into 5 groups. Sixteen canine prostates were treated with the third-generation transrectal HIFU device (Sonablate-500^™). Transrectal ultrasound images of the prostate and prostatic urethra were observed preoperatively and postoperatively. Serial study was performed 30 min, 30 days, 60 days and 180 days after the therapy. The rectum, periprostatic tissues, and prostate were excised en bloc and the tissues were fixed for gross and histological analysis. Our results showed that the average maximal diameter of prostatic urethra was 0.59±0.11 cm before the operation and 2.57±0.98 cm 60 days after the operation. The volume of prostate was 6.5±3.12 cm³ before the treatment while the volume was 4.13±0.23 cm³ 60 days after the treatment and the differences were statistically significant (P<0.05). Histologically, there was a clear demarcation between the necrotic area of the treated tissues and the unaffected surrounding tissues. All the necrotic tissues in the targeted zone broke off and the prostatic urethra became cavitary 60 days later. The more frequent complications were urinary retention and frequency and hematuria. No rectal injury occurred during the treatment. It is concluded that the third-generation transrectal HIFU is capable of destroying prostatic tissue, substantially increasing the width of the prostatic urethra without causing injury to the adjacent tissues. The risk of postoperative complications associated with HIFU was low. HIFU may become a safe, effective and minimally invasive alternative for the treatment of prostatic diseases.

To observe the efficacy and safety of mini-percutaneous nephrolithotomy with Neodymium: Yttrium Aluminum Garnet (Nd-YAG) laser in the treatment for upper urinary tract stones, from December 2005 to September 2006, 31 patients with renal stones, 15 patients with ureteral stones and 7 patients with renal and ureteral stones underwent mini-percutaneous nephrolithotomy with Nd-YAG laser by combination of rigid ureteroscope and flexible ureteroscope under B-ultrasound guidance. Clinical data including operation time, lithotripsy time, complications and stone-free rate were analyzed retrospectively. Our study showed that the percutaneous renal access (F14–F18) was successfully established under B-ultrasound guidance in all cases. Immediate phase: lithotripsy was performed in 47 cases through single tract, and in one case through two tracts. Delayed phase II lithotripsy was done in 5 cases of renal stones. Operation time ranged from 55 to 180 min with an average time of 100±15 min. Lithotripsy time was from 25 to 135 min and the average lithotripsy time was 65±11 min. No severe complications occurred in our series. Complex renal stones were cleared in 34 of 38 cases (89.5%). All ureteral stones were completely removed in 15 cases (100%). It was concluded that mini-percutaneous nephrolithotomy with Nd:YAG laser for the treatment of upper urinary tract stones by combination of rigid ureteroscope and flexible ureteroscope has the advantages of effectiveness, mini-invasion, shorter operative time and safety.

This study examined the expression and distribution of angiopoietin-1/-2 (Ang-1/-2) in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohisto-chemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups: control group, D2 group (2 days after pregnancy), D4 group (4 days after pregnancy), D6 group (6 days after pregnancy) and D8 group (8 days after pregnancy), each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups (P<0.01). Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy (day 2), and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice (P<0.01). In situ hybridization showed Ang-1 mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 (P<0.01). It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

To investigate the role of toll-like receptor 9 (TLR9) in the pathogenesis of lichen planus, the expressions of TLR9 and its mRNA in the lesional skin of lichen planus were detected by immunohistochemical technique (SP) and RT-PCR. As control, normal skin of healthy volunteers was also tested. The immunohistochemical study showed that the expression of TLR9 in the lesional skin of lichen planus was significantly higher than that in the normal controls. The results of RT-PCR showed that both skin lesions and normal controls had TLR9 expression. In skin lesions, the expression level of TLR9 mRNA was 1.6075±0.0930, which was significantly higher than that in normal controls (P<0.001). These findings indicated that up-regulated expression of TLR9 and its mRNA might be involved in the pathogenesis of lichen planus.

To study the expression of p63 and cyclooxygenase-2 (cox-2) in skin tumors and evaluate the correlation between p63 and cox-2, the expressions of cox-2 and p63 were measured by streptavidin-peroxidase complex immunohistochemical technique in 17 cases of skin squamous cell carcinoma (SCC), 19 cases of Bowen’s disease(Bowen), 11 cases of actinic keratosis(AK), 12 cases of seborreic keratosis(SK) and 13 specimens of normal skin. Our results showed that the expression of p63 in skin squamous cell carcinoma, Bowen’s disease and actinic keratosis were significantly higher than that in seborreic keratosis, while the expression of p63 in seborreic keratosis was significantly higher than that in normal skin. The expression of cox-2 in skin squamous cell carcinoma, Bowen’s disease and actinic keratosis were significantly higher than that in seborreic keratosis, while no statistical difference was noted in the expression of cox-2 between seborreic keratosis and normal skin. Cox-2 expression was positively correlated with the high p63 expression in malignant skin tumors. The increased expression of cox-2 and p63 may play an important role in the development of skin tumors and work synergetically in malignant skin tumors.

To compare the therapeutic effects of three different anti-fungal drugs (i.e., terbinafine, fluconazole and intraconazole) in the treatment of experimental vaginitis caused by Candida albicans (C. albicans) in mice, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animal had been pretreated with estradiol. Mice were divided at random into different groups and then respectively treated with terbinafine, fluconazole and intraconazole given by gastrogavage. The burden of the fungus in the vaginal lavage fluids in the mice of the different groups was measured dynamically at different time points after the beginning of the drug treatment. The fungal burdens in the vaginal lavage fluids taken at different time points from the mice treated with terbinafine were significantly higher than those taken at corresponding time points from mice treated with fluconazole or itraconazole (P<0.01). The fungal burdens in the vaginal lavage fluids taken from mice 1 week after the beginning of the treatment with terbinafine remained at a relatively high level. A dramatic drop in the fungal burden was noted in the vaginal lavage fluids taken on the 2nd day of the treatment from mice treated with itraconazole or fluconazole group and the fungal burden on the 3rd day of the treatment in these mice were at a very low level, suggesting that fluconazole or itraconazole were highly effective for the treatment. However, the difference in the therapeutic effect between the two drugs was not significant (P>0.05). Itraconazole or fluconazole, but not terbinafine, is very effective for the treatment of fungal vaginitis caused by C. albicans in mice.

In this study nine cases of thallium poisoning in a series of homicidal poisoning were analyzed in order to provide more information concerning thallium poisoning. It was found that the most common clinical feature of thallium poisoning was peripheral neuropathy and paraesthesia was more common than amyasthenia. Understanding of these clinical characteristics of thallium poisoning was helpful to early identification and differential diagnosis. Since the early administration of Prussian Blue, as a specific antidote for thallium poisoning, can substantially improve the prognosis, it is of great importance to establish a correct and early diagnosis.

To investigate the high-risk factors for newborn hearing loss and to provide information for preventing the development of hearing loss and delaying its progression, from May 2003 to June 2006, neonates who failed to pass the universal newborn hearing screening (UNHS) were referred to Jinan Newborn Hearing Screening and Rehabilitation Center from 7 newborn hearing screening centers in seven cities of Shandong province. One-to-one pair-matched case-control method was employed for statistical analysis of the basic features of definitely identified cases. High-risk factors relating to the bilateral hearing loss were evaluated by univariate and multivariate Logistic regression analysis. Our results revealed that 721 transferred newborns who didn’t pass the hearing screening received audiological and medical evaluation and 367 were confirmed to have hearing loss. Of them, 177 neonates with hearing loss who met the matching requirements were included in the study as subjects. Univariate analysis showed that high-risk factors related to hearing loss incuded age of father, education backgrounds of parents, parity, birth weight, gestational weeks, craniofacial deformity, history of receiving treatment in neonatal intensive care unit (NICU), neonatal disease, family history of otopathy and family history of congenital hearing loss. Multivariate Logistic regression analysis revealed that 4 independent risk factors were related to bilateral hearing loss, including parity (OR=16.285, 95% CI 3.379–78.481), neonatal disease (OR=34.968, 95% CI 2.720–449.534), family history of congenital hearing loss (OR=69.488, 95% CI 4.417–1093.300) and birth weight (OR=0.241, 95% CI 0.090–0.648). It is concluded that parity, neonatal disease and family history of hearing loss are the promoting factors of bilateral hearing loss in neonates and appropriate intervention measures should be taken to deal with the risk factors.