Objective To observe the expressing features of EGF and PCNA and study their roles during the development of human fetal testis. Methods The positive rates of EGF and PCNA in Leydig cells,Sertoli cells and germ cells of human fetal testis from 12th to 32th week were examined by SP immunocytochemical technique. Results The positive cells of EGF were not found in 12th week,and were found in Leydig cells from 16th to 32th week,the peak of expression was observed in 16th week.The positive staining was orange or brown and were scattered within the cytoplasm,the number of positive cells decreased gradually with the increasing of the fetal ages (P

Objective To investigate the morphological changes and expression of nerve growth factor(NGF) in submandibular gland of C57BL/ksj\|db/db mutant diabetic mice. Methods Animals were C57BL/ksj\|db/+m mutant diabetic mice which were imported from Japan.The pure Zygote,that is db/db mouse,displays obesity and high blood glucose over 10mmol/L.The test groups were 3,4,6,8,10 month\|old db/db mice and the age\|matched db/+m mice as control.Immunohistochemistry technology was applied in this study. Results In db/db diabetic mice,submandibular gland atrophied and took on the significant morphological changes in acinar,duct and granular convoluted tubule.The NGF Immunopositive cells in submandibular gland were significantly less than the age\|matched control animals and showed a descend tendency with the progression of diabetes millitus.Conclusion\ The decreased expression of NGF demonstrates NGF synthesis and secretion in granular convoluted tubule of submandibular gland was decreased.The lower NGF levels may be associated with development of diabetic neuropathy.\;[

Objective In order to investigate the roles of hIGF\|1 in treatment of diabetes mellitus and diabetic syndromes,the gene of human insulin like growth factor type Ⅰ(IGF\|1) was cloned and constructed into eukaryotic expression vector,then the expression and activity were determined. Methods Total cellular RNA of human fetal liver was abstracted and the RT\|PCR amplification of the cDNA fragment was performed.The fragment was cloned into pUCM\|T vector and sequenced.The eukaryiotic expression vector was recombined and transfected into fibroblast cell line,COS\|7.The expression of hIGF\|1 was examined by in situ hybridization and immunohistochemistry.The effect of hIGF\|1 on cultured islet cells was observed by glucose\|stimulated insulin release assay. Results The cDNA fragment of 710bp with additional Kozak sequence was amplified by RT\|PCR.Eukaryiotic expression vector pCI\|neo/hIGF\|1 was constructed and IGF\|1 gene expressed in COS\|7.The biological activity of hIGF\|1 was proved by increasing inslin secretion from islet cells.Conclusion\ The newly constructed vector,pCI\|neo/hIGF\|1 could be transfected into COS7 cells and its expressed product showed to have the biology activity of hIGF\|1.\;[

Objective In order to investigate the effect of nutrition and induction of Sertoli cell on cultured neural stem cells(NSCs) from embryonic mesemcephalon in the rat. Methods Sertoli cells from the testis and the neural precursors(NPs) from the ventral mesencephalon in embryonic SD rat(E13) were co-cultured.The detections of immunoreactivities of nestin,?-Ⅲ tubulin and tyrosine hydroxylase(TH) were made by immunocytochemistry after 5, 7, 10 and 14 days in vitro. Results The number of NPs with nestin-positive was decreasing along the time of the co-culture,while the ?-Ⅲ tubulin-positive cells and TH-positive cells were increasing.The number of ?-Ⅲ tubulin-positive cells was quite high at the 7th day and the TH-positive cells were at the highest level at the 10th day in vitro.At any stage,the numbers of TH-positive cells were higher in the co-culture,compared with those in the mono-culture.Conclusion The neural precursors from the ventral mesencephalon in embryonic rat can be directly induced to differentiate into the dopaminergic progenitors by co-cultured with the Sertoli cells.

Objective To study the influence of 4-aminopyridine(4-AP)on proliferation,production,and apoptosis through inhibiting voltage-gated K+channel(Kv)in ovarian luteinized granulosa cells.nethods Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programme.The cultured granulosa cells were divided into 4 groups:blank group,4-AP treated group,human chorionic gonadotropin(hCG)-induced group and hCG+4-AP cotreated group.The final concentrations of hCG and 4-AP were 1250 U/L and 5 nmol/L respectively.The progesterone production WaS detected by the chemoluminescence method.The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by RT.PCR The influence on the early apoptosis of gTanulosa cells bv 4-AP was observed by flow eytometry.Cellular caSpage-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium(MTT)method.Results(1)Kv mRNA wag expressed in granulosa cell.(2)The progesterone production64),(206±32),(1991±172)and(763±79)nmol/L,respectively after24 hours culture.Exposure of the(3)The flow cytometry analysis and the cellular caapase-3 A405 showed that 4-AP increased the percentage ofearly phase apoptosis(P<0.01):4-AP treated group VS blank group[(40±5)%and 0.049 ±0.009]VS[(17±4)% and 0.029±0.008],hCG+4-AP CO-treated group VS hCG-induced group[(25±4)%and0.039 ±0.0081 VS[(15±3)%and 0.022 ±0.007].(4)24 hours after treated with 4-AP and hCG,theinhibitory rate of cultured granulosa cells of 4-AP treated group was higher than the blank group(19.7%VS0).and that of hCG+4-AP co-treated group was obviously higher than hCG-induced group(34.6% VS O,P<0.01).Conclusions The voltage-gated K+ channels expressed by ovarian luteinized granulosa cellplay an important role in cell proliferation,production,and apoptosis.4-AP may inhibit differentiation ofprogesterone in granulosa ceHs through the inhibition of proliferation and induction of apoptosis.

Objective To observe whether the expression of Annexin A5(ANXA5) changes in human uterine cervical squamous cell carcinomas(UCSCC).Methods 25 UCSCC tissues and 15 normal human uterine cervical tissues were collected.Each sample was lysed in lysis buffer.Whole protein of the supernatant was estimated by BCA-100 Protein Quantitative Analysis Kit.The expressions of ANXA5 in UCSCCs and normal uterine cervical tissues were detected respectively with Western blotting.To further ensure the expression of ANXA5 in UCSCCs,another 45 UCSCC specimens and 20 normal cervical tissues were collected.ANXA5 expression was detected by in situ hybridization and immunohistochemistry respectively.Results The staining of ANXA5 band with Western blotting in UCSCCs was much heavier than that in normal uterine cervical tissues and the expression of ANXA5 was found much higher in UCSCCs by immunohistochemistry and in situ hybridization.Conclusion Expression of ANXA5 was up-regulated in human UCSCCs.There's some relationship between uterine cervical squamous cell carcinoma and ANXA5 protein.

Objective To investigate the effect of Desert Hedgehog on direct differentiation of neural progenitor cells(NPCs) cultured from embryonic mesencephalon in the rats.Methods We infected DHH into COS7,NIH/3T3 and 9L cells,and detected the expression of DHH in the cells with immunofluorescence,real-time PCR and Western blot.All of the three cells were co-cultured with NPCs isolated from ventral mesencephalon in embryonic SD rats(E13.5) respectively.Immunoreactivities of tyrosine hydroxylase(TH) was detected by immunocytochemistry after 10 days.Results The expression of DHH in COS7,NIH/3T3 and 9L cells was remarkably detected,but few TH-positive cells were found in the three co-cultral systems at the 10th day.Conclusion The protein derived from DHH itself does not show any inductive effect on the differentiation of NPCs to the dopaminergic neurons in vitro.

Objective To explore if bacillus bifidus relieve CPFX-induced testosterone reduction in mouse testes.Methods Twenty-four male mices were divided into 4 groups, then administered saline for 6 days (Sal6 group), CPFX for 6 days (A6 group), CPFX for 6 days followed by bifidobacteria treatment for the next 6 days (A6+P6 group), CPFX for 6 days and then saline for the next 6 days (A6+Sal6 group).We detected serum levels of testosterone by RIA, as well as levels of steroidogenic enzymes mRNA [cholesterol side-chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR)] and NF-E2-related factor2 (Nrf2) mRNA in testes by real-time PCR, Nrf2, heme oxygenase-1 (HO-1), and 4-hydroxy-2-nonenal (4-HNE) by Western blot and4-HNE by Immunohistochemistry.Results The A6 group had significantly lower serum testosterone levels compared with the Sal6 group (P<0.001), the A6+P6 group had significantly higher compared with the A6 (P<0.001) and A6+Sal6 groups (P<0.01).The A6 group had significantly lower StAR mRNA compared with the Sal6 group (P<0.001), the A6+P6 group had significantly higher level compared with the A6 (P<0.01) and A6+Sal6 groups (P<0.01).The A6 group had significantly lower P450scc mRNA as compared with the Sal6 group (P<0.001), the A6+P6 group had significantly higher compared with the A6 (P<0.001) and A6+Sal6 groups (P<0.05).The A6 group had significantly lower Nrf2 compared with the Sal6 group (P<0.001), the A6+P6 group had significantly higher compared with the A6(P<0.01) and A6+Sal6 groups (P<0.05).The A6 group higher 4-HNE expression compared with the Sal6 group, the A6+P6 group had significantly lower compared with the A6 (P<0.01) and A6+Sal6 groups (P<0.05).Conclusions Bifidobacteria the reduction of CPFX-induced testosterone reduction, and these effects may potentially explained by Nrf2 inflammatory signaling pathway.

Objective To investigate the expression of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor-4 ( FGFR-4 ) in the papillary thyroid carcinomas ( PTC ) and clinical significance . Methods Immunohistochemistry and Western blotting for the expression of FGF-2 and FGFR-4 were performed in 89 cases of PTC and 30 cases of normal thyroid tissues ( NTT) adjacent to the tumors .Results Immunohistochemistry results showed that , FGF-2 and FGFR-4 expressions were high in thyroid carcinoma (P<0.01,P<0.01) in contrast to that in the normal thyroid tissues, and the difference was statistically significant;There was a positive linear correlation between expressions of FGF-2 and FGFR-4 and lymph node metastasis (χ2 =14.798,P<0.01;χ2 =7.27,P<0.01)and differentiation degree (χ2=13.824,P<0.01;χ2 =16.921, P<0.01) in papillary thyroid carcinoma ,while there was no difference in gender ,age and tumor size(P>0.05).Analyzed by Western blotting technique ,FGF-2 and FGFR-4 expressions in thyroid carcinoma were significantly higher than that in normal tissue ,with decrease of cancer degree of tissue differentiation and significantly up regulated expression (P<0.05).Expressions of FGF-2 and FGFR-4 were in a positive linear correlation in the disease (rs=0.434,P<0.01).Conclusion The expressions of FGF-2 and FGFR-4 are correlated with papillary thyroid cancer and they participated in the process of invasion and metastasis , both of which have a positive synergistic effect .The degree of malignancy and biological behavior are meaningful and comprehensive indicators ,which provide a theoretical basis for the subsequent experimental studies of cellular and molecular biology .