[Differentially expressed genes in diabetes-induced embryopathy].

To determine molecular mechanism in hyperglycemia induced congenital neural tube defects, yolk sac cells were harvested at gestational day 12 from streptozotocin (STZ) -induced diabetic rats with congenital neural tube defects in offspring, STZ-induced diabetic rats without neural tube defects and normal control group. We analyzed gene expression profiles in yolk sac cells using a DNA microarray technique. Changes in apoptotic and MAP Kinase signaling pathways were detected by Western blotting analyses. Comparison of genes in yolk sac cells with a total of 1 200 genes in the control cells, 79 genes differently expressed between the two groups were detected. Forty-two of them were up-regulated and 37 were down-regulated. There was strong characteristic apoptotic DNA ladder in yolk sac cells in embryopathic offspring from experimentally-induced diabetic rats. The activity of ERK1/2 was dramatically decreased and the activity of JNK1/2 was significantly increased. Differentially expressed genes, MAP Kinase, and apoptotic signal pathways play very important roles in hyperglycemia induced neural tube defects. We hope that these could provide useful hallmark to rapid identification of early diabetic embryopathy.

Survivin gene RNA interference inhibits proliferation, induces apoptosis, and enhances radiosensitivity in HeLa cells.

OBJECTIVE: Survivin is a new member of the inhibitors of apoptosis (IAPs) family. It is upregulated in various malignancies including human cervical carcinomas. Reduction of this molecule has resulted in chemosensitization, but it is uncertain whether it can lead to radiosensitization. We observed the effect of survivin gene RNA interference (RNAi) on the proliferation, apoptosis, and radiosensitivity of the human cervical carcinoma cells HeLa. STUDY DESIGN: Human cervical carcinoma cells (HeLa) were transfected with the specific siRNA expression vector (pSilencer2.1-s2) designed to target survivin mRNA. A corresponding site-mutated vector was constructed as a negative control (pSilencer2.1-NC). The expression of survivin mRNA and its protein among the stable transfected cells and the untransfected ones was detected by semi-quantitative RT-PCR and Western blotting respectively. The cell growth was examined by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle distribution and cell apoptosis were measured by flow cytometry. The changes in cell radiosensitivity were observed by clonogenic survival assay. RESULTS: Three stable transfected cell lines: HeLa-s2 (with pSilencer2.1-s2), HeLa-NC (with pSilencer2.1-NC), and HeLa-U6 neo (with empty vector pSilencer2.1-U6 neo) were established. The expression levels of survivin gene mRNA and protein in HeLa-s2 were significantly lower than in HeLa-NC, HeLa-U6 neo, and those untransfected HeLa cells. The expression inhibitory rates were 62.8% and 60.1%. The cell proliferation of HeLa-s2 was inhibited, and the highest inhibitory rate was 57.8+/-2.1%. The changes in cell cycle distribution in HeLa-s2 compared with the other three cell lines were obvious, many cells were blocked in the G(0)/G(1) phase 72.7+/-3.1% (P<0.05), reduced sharply in the G(2)/M phase (5.1+/-2.9)% (P<0.05), and also the apoptotic rate was 29.2+/-1.4%, obviously increasing (P<0.05). At the same dose of radiation, the cloning efficiency of HeLa-s2 declined notably (P<0.05); the cell survival curve showed a significant decrease in D(0) and D(q), which were 3.15 and 1.21, respectively (P<0.05), and the radiation enhancement ratios were 2.01 (a ratio of D(0)) and 1.77 (a ratio of D(q)). CONCLUSIONS: Survivin gene RNAi not only could inhibit the proliferation of human cervical carcinoma cells (HeLa), but also could significantly enhance the radiosensitivity of those cells through the reduction of its mRNA and protein. Therefore, an RNAi-targeted survivin gene strategy would be a potential approach to radiosensitization therapy in human cervical carcinomas.

[Influence of survivin gene repression by RNA interference on the radiosensitivity and chemosensitivity to cisplatin of cervical cancer cell HeLa].

OBJECTIVE: To observe the effect of survivin gene repression by RNA interference (RNAi) on the radiosensitivity and the chemosensitivity to cisplatin of cervical cancer cell HeLa. METHODS: The recombined eukaryotic expression plasmid pSilencer2.1-s2 containing human survivin gene small interference RNA (siRNA) was transfected into human cervical cancer cell line HeLa by using lipofectamine 2000. The expression of survivin gene mRNA and its protein was detected by semi-quantitative RT-PCR and western blot respectively. The changes of caspase-3 activity was assessed by kinase activity test. Cell apoptosis was examined by flow cytometry. The changes of cell radiosensitivity was observed by plate clone formation assay. Cell viability was examined by methyl thiazolyl tetrazolium (MTT) assay and 50% inhibitive concentration (IC(50)) of cisplatin was examined also. RESULTS: Compared with the cells which were transfected with negative control plasmid (HeLa-NC), pure plasmid (HeLa-U6 neo) and un-transfected cells (HeLa), the expression level of survivin gene mRNA and protein declined evidently in the cells transfected with pSilencer2.1-s2 plasmid (HeLa-s2), the expression inhibitory rates were (62.8 +/- 0.3)% and (60.1 +/- 0.5)%; the caspase-3 activity was enhanced and A(405) was 1.261 +/- 0.043 (P < 0.05); the apoptotic rate was increased obviously (29.23 +/- 1.41)% (P < 0.05). At the same dose of radiation, clone formation rate declined significantly (P < 0.05); at the same concentration of cisplatin, cell viability declined sharply and the IC(50) of cisplatin was (0.873 +/- 0.021) microg/ml (P < 0.05). CONCLUSION: Survivin gene repression by RNAi can enhance caspase-3 activity, induce cell apoptosis, significantly increase the radiosensitivity and chemosensitivity to cisplatin in human cervical cancer cell line HeLa.

[MAP kinase signal pathway in hyperglycemia-induced congenital neural tube defects].

The aim of the present study was to determine molecular mechanism in hyperglycemia-induced congenital neural tube defects and the its potential pharmacologic rescuing agents. In order to explore these questions, six study groups of Sprague-Dawley rats were employed: Group 1 was normal control rats with normal diet; group 2 represented streptozotocin (STZ) -induced diabetic rats with congenital neural tube defects in offspring; group 3 included STZ-induced diabetic rats with normal offspring; groups 4,5 and 6 included rats exposed to the same STZ-induced diabetic condition, but receiving daily oral supplementation of 80 microg/mL of the sodium salt of arachidonic acid (AA), 400mg of vitamin E and a cocktail of a polyunsaturated fatty acid (safflower oil) plus an antioxidant ( vitamin E) respectively. Yolk sac cells were harvested at gestational day 12 from each rat group. Changes in MAPK signaling pathways were detected by western blot analysis using special antibodies directed against phosphorylated forms of extracellular signal regulated kinase (ERK), Jun N-terminal/stress-activated protein kinase (JNK/SAPK). Furthermore, activity of RAF-1, an upstream kinase in ERK1/2 signaling cascade, was evaluated by immunoprecipitation assay. The results showed that in yolk sac cells in embryopathic offspring from experimentally-induced diabetic rats, activities of ERK1/2 were dramatically decreased (group 2). Consisted with these observation, reduction in RAF-1 kinase activity could be discerned in these diabetic yolk sac cells. In contrast, activities of JNK1/2 were significantly increased in yolk sac cells of group 2. Under rescuing circumstance,activations of ERK1/2 and RAF-1 were increased, and JNK1/2 were decreased. MAP kinase signal pathway plays a very important role in hyperglycemia induced neural tube defects. The supplementation of polyunsaturated fatty acid arachidonic acid, and antioxidant vitamin E rescued conceptuses from diabetic embryopathy by triggering a restoration of normal membrane signaling pathways.

[Effect of huangqi and dangshen extraction on angiogenesis in Matrigel implant of mice].

AIM: To study the inhibitory effect of huangqi and dangshen extraction (SQ) on angiogenesis induced by b-FGF. METHODS: Matrigel implant assay was used. Matrigel(500 microL) containing b-FGF and heparin was injected subcutaneously into the abdomens of mice and harvested 5 d later. The amount of hemoglobin and micro-vascular area present in the implant were measured and compared. The mice were given different dosage of SQ (experimental group) or the same volume of glucose (vehicle group) once a day by intraperitoneal injection. Inhibitory experiment started 3 d before Matrigel implant and continued until the end of study. RESULTS: SQ in lower dosage (< or = 50% V/V) increased hemoglobin content and micro-vascular area in Matrigel implant while SQ in higher dosage (> or = 60%, V/V) reduced hemoglobin content and micro-vascular area in Matrigel implant. The effect of enhance ment and inhibition was in a limited concentration-effect manner. CONCLUSION: SQ in different dosage has different effects on angiogenesis. We should use different dosage in different purpose.

[Inhibitory effect of huangqi and dangshen extraction with pacilitaxel on metastasis and angiogenesis on mouse Lewis lung carcinoma model].

AIM: To study enhance effect of huangqi and dangshen extraction (Shenqi) on pacilitaxel inhibitory metastasis and angiogenesis on mouse Lewis lung carcinoma model. METHODS: Lewis lung carcinoma cells were inoculated into right hind footpad of C57BL/6 mice. Six hour after tumor inoculated, the mice were randomly divided into 3 groups.Shenqi (paclitaxel plus Shenqi) or paclitaxel was intraperitoneally injected in two group since the second day of the establishment of animal model. The third group simply administered with normal saline was set as placebo-control. Tumor volume, quantitation of microvessel density (MVD) in inoculated tumor, the number of metastasis in the lungs and survival analysis were compared in 3 groups. RESULTS: Paclitaxel plus Shenqi can effectively reduced MVD in inoculated tumor and the number of lung metastasis as compared with other two group (P<0.05). The survival time of Shenqi group was also significantly longer (P<0.05). Tumor volume was no statistical difference in three group (P>0.05). CONCLUSION: Shenqi can amplify the paclitaxel effect of anti-angiogenesis and anti-metastasis, enhances the survival time of mice bearing LLC, might has possible therapeutic applications in the treatment of lung cancer.

[The expression of acetyl-heparanase, laminin and laminin receptor and their role in the metastasis of ovarian cancer].

AIM: To explore the expressions of acetyl-heparanase mRNA, laminin ( LN) and laminin receptor ( LR) in 50 ovarian carcinoma, 33 ovarian carcinoma with lymph node metastasis, and 10 serous ovarian cystadenoma as well as their role in the metastasis of ovarian cancer. METHODS: The transcription level of acetyl-heparanase mRNA, expressions of LN and LR were detected by in situ hibridization and immunohistochemical staining, respectively. RESULTS: The transcription level of acetyl-heparanase mRNA in ovarian carcinoma tissue and metastatic lymph nodes increased significantly, but its expression in primary focus was notably higher than that in metastatic lymph nodes (P < 0. 05 ). There was low expression of acetyl-heparanase mRNA in serous ovarian cystadenoma. The expression of acetyl-heparanase mRNA in malignant and benign tumor tissues had markedly difference (P < 0. 01 ). Expressions LN in both tissues mentioned above decreased while LR expression was high. The expression of acetyl-heparanase mRNA was negative correlation with that of LN, while positive with that of LR. CONCLUSION: The correlation among expressions of acetyl-heparanase mRNA, LN and LR suggests that heparanase is involved in the growth, invasion and metastasis of ovarian carcinoma.

[Effect of insulin on apoptosis of cultured human trophoblast cells and its mechanism].

AIM: To explore the effect of insulin on apoptosis of cultured human trophoblast cells and its possible mechanism. METHODS: Human trophoblast cells from early pregnancy women were cultured and divided into 3 groups; normal control group; H2O2, treatment group and insulin plus H2O2 treatment group. H2O2 was used to induce apoptosis of trophoblasts cells. Apoptotic rate was detected by flow cytometry. The effects of insulin on Bcl-2 expression and caspase-3 activity were also detected. RESULTS: H2O2 might induce apoptosis of trophoblast cells and typical morphological features of apoptotic cells was observed under electron microscope. Flow cytometry detection exhibited that insulin could reduce markedly H2O2-induced apoptotic rate of trophoblasts cells (P < 0. 01). Bcl-2 expression rate inH,O, treatment group was significantly lower than that in control group (P < 0. 01), while caspase-3 activity was distinctly higher than that in control group (P < 0. 01). CONCLUSION: Insulin could inhibit apoptosis of human trophoblasts cells induced by H2O2, which be may through decreased caspase-3 activity and increased Bcl-2 protein expression.

[Development and application of an indirect immunofluorescence assay for the detection of serum immunoglobulin G to human herpesvirus 6].

AIM: To establish an indirect immunofluorescence assay(IFA) for detection of serum IgG antibody against human herpesvirus 6(HHV-6). METHODS: Cord blood mononuclear cells infected by HHV-6 strain CN(5) were used to prepare cell antigen smear, so as to establish an IFA and make an epidemical investigation on serum specimens of child-bearing age, women. RESULTS: The specificity of the IFA for HHV-6 was confirmed by absorption assay(test). The IFA detection showed that in serum specimens from 116 cases of child-bearing age, women, the positive rate of anti-HHV-6 IgG was 72.4% and geometric mean titer(GMT) was 1:61. The positive rate and GMT of serum anti-HHV-6 IgG had no differences between pregnant women and unpregnant women, neither among pregnant women at different pregnant stages. CONCLUSION: A specific IFA has been developed successfully for epidemical investigation of HHV-6 infection rate in child-bearing women.

[Experimental study of anti-VEGF hairpin ribozyme gene inhibiting expression of VEGF and proliferation of ovarian cancer cells].

BACKGROUND & OBJECTIVE: Growth of solid tumor metastases are critically dependent on angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been identified as one of the most potent inducers of tumor associated angiogenesis, studies have shown that VEGF plays an important role in angiogenesis which is associated with epithelial ovarian cancer. Until now, many strategies for gene therapy have been developed. Among them is Ribozyme-based therapeutics for cancer which might be devised to inhibit tumor growth or prevent metastases. Angiogenesis is required for sustained tumor growth, making the VEGF pathway another promising target for either small molecule or nucleic acid-based therapeutics. Little is known about the role of VEGF in ovarian tumorigenecity. We propose to block the autocrine and/or paracrine pathway of VEGF in ovarian cancer using anti-VEGF hairpin ribozyme gene to see whether the growth of tumor cells could be inhibited and to further exploit its mechanisms. METHODS: Anti-VEGF hairpin ribozyme gene eukaryotic expression vector was introduced into ovarian cancer SKOV3 cells by lipofectin mediation and positive clones were screened by G418; Ribozyme expression was confirmed by RNA dot blot; The VEGF expression of SKOV3 cells before or after transfection were detected by immunohistochemical and immunofluorescence and flow cytometer immunofluorescence methods, MTT, colony forming, soft agar colony forming, and FCM were used to observe the effect of proliferation to ovarian cancer cells. RESULTS: VEGF expression decreased distinctly in SKOV3-RZ cells. The growth of transfected SKOV3-RZ cells were slower, The average colony forming efficiency and soft agar colony forming efficiency of SKOV3-RZ cells(12.7 +/- 1.4 and 9.4 +/- 2.0, respectively) decreased distinctly (P < 0.001). The SKOV3-RZ cells of G1 stage increased(P < 0.01), the SKOV3-RZ cells of S stage were reduced(P < 0.01). CONCLUSIONS: Anti-VEGF hairpin ribozyme gene can inhibit the proliferation of ovarian cancer SKOV3 cells. This provides a experimental basis for cure human ovarian cancer with antiangiogenesis method.