Objective To explore specific gene expression for regulating meiosis of germ cells during spermatogenesis of rat testis Materials & Methods Male SD rats, aged 1, 3 and 8 weeks, were observed in this study. The ...Objective To explore specific gene expression for regulating meiosis of germ cells during spermatogenesis of rat testis Materials & Methods Male SD rats, aged 1, 3 and 8 weeks, were observed in this study. The methods of morphological observation on testicular tissues embedded by resin and mRNA differential display (DDRT PCR) were combined to obtain specific mRNA expression gene fragments during the testicular development. Reverse dot blot hybridization was operated to further screen the positive differential DNA fragments. The positive DNA segments were sub cloned in pGEM T Easy vector and transformed into the competent E coli 109 straint. Northern blot analysis and in situ hybridization were also carried out for identifying tissue specific expression as well as cell specific expression DNA fragments. To screen λ ZAP II rat testicular gene library was searched for the original gene. Results Eighty two differential cDNA fragments were obtained through primary DDRT PCR, among which 40 differential cDNA fragments were selected for further screening with reverse dot blot hybridization. After the reverse dot blot hybridization, 12 primary differential DNA fragments were obtained. The size of DNA fragments ranged from 250 to 500 bp. The in situ hybridization of the testicular tissue showed that a specific DNA fragment derived from 8 week old rat testis, named CG14, was hybridized in adult rat testicular section, in which the positive nucleic acid signals were distributed specifically in the primary spermatocytes. Another DNA fragment derived from 1 week old rat testis, named AA11, was hybridized specifically in Sertoli cell of 1 week old rat testis. Northern blot hybridization with [α 32 P] dCTP labeled CG14 probe, including cardiac, liver, kidney, brain, testis, and epididymis tissue mRNAs of rat, showed that an mRNA specific hybridization band, size of 1.258 kb, was found in testis tissue and size of 1.531 kb of another hybridization band present in epididymis tissue. The CG14 DNA specific gene fragment existed in the λ ZAP II testis gene library. Conclusion 1. The DDRT PCR technique is a convenient tool to find the specific expression gene during spermatogenesis. 2. The CG14 DNA fragment was expressed significantly in rat testicular tissue, weakly expressed in the epididymis tissue of rat, but not found in cardiac, liver, kidney, and brain tissues. 3. The CG14 DNA fragment was specifically expressed in the primary spermatocytes and might be associated with the meiosis of the primary spermatocyte during spermatogenesis of rat. 4. The CG14 DNA fragment existed in the λ ZAP II testis gene library.展开更多
Objective To investigate the role of fibronectin(FN) receptor α 5β 1 in liver fibrosis Methods Northern blot analysis and immuno^histochemical techniques were used to observe changes in the expression of FN an...Objective To investigate the role of fibronectin(FN) receptor α 5β 1 in liver fibrosis Methods Northern blot analysis and immuno^histochemical techniques were used to observe changes in the expression of FN and FN receptor α 5β 1 on hepatic stellate cells (HSCs) in vivo and in vitro of rat liver fibrosis induced by CCl 4 Results (1) α 5β 1 was mainly detected in the endothelia and some of the desmin(DM) positive cells of the sinusoids in normal rat liver The expression of α 5β 1 of DM positive cells detected by immunohistochemistry reached its peak at the 10th week of the experiment The changes in FN expression were similar to that of α 5β 1 (2) The expression of FN, α 5 and β 1 mRNAs in the experimental group was remarkably increased especially at the 6th week, compared to that of normal liver specimens The expression of the three mRNAs of HSCs in vitro isolated from the experimental group (6 weeks) was higher than those from normal liver (3) The expression of FN, α 5 and β 1 mRNAs increased in normal rat HSCs after treatment with transforming growth factor β 1 (TGF β 1) for 2 hours After 4 hours of treatment, the expression of the three mRNAs fell to levels similar to the control group Immunocytochemistry revealed that the expression of α 5β 1 of HSCs reached its peak after 4 hours of treatment with TGF β 1 and dropped to normal 2 hours later Conclusion These data suggest that HSCs normally express FN receptor α 5β 1 The activation of HSCs during liver fibrogenesis leads to an increase of FN, α 5 and β 1 mRNA expression The expression of FN and α 5β 1 of HSCs in vitro is up regulated by TFG α 5β 1 The detection of gene transcription of FN and its receptor by Northern blot analysis suggests the activation and proliferation of HSCs and thereby provides a sensitive signal of liver fibrosis展开更多
文摘Objective To explore specific gene expression for regulating meiosis of germ cells during spermatogenesis of rat testis Materials & Methods Male SD rats, aged 1, 3 and 8 weeks, were observed in this study. The methods of morphological observation on testicular tissues embedded by resin and mRNA differential display (DDRT PCR) were combined to obtain specific mRNA expression gene fragments during the testicular development. Reverse dot blot hybridization was operated to further screen the positive differential DNA fragments. The positive DNA segments were sub cloned in pGEM T Easy vector and transformed into the competent E coli 109 straint. Northern blot analysis and in situ hybridization were also carried out for identifying tissue specific expression as well as cell specific expression DNA fragments. To screen λ ZAP II rat testicular gene library was searched for the original gene. Results Eighty two differential cDNA fragments were obtained through primary DDRT PCR, among which 40 differential cDNA fragments were selected for further screening with reverse dot blot hybridization. After the reverse dot blot hybridization, 12 primary differential DNA fragments were obtained. The size of DNA fragments ranged from 250 to 500 bp. The in situ hybridization of the testicular tissue showed that a specific DNA fragment derived from 8 week old rat testis, named CG14, was hybridized in adult rat testicular section, in which the positive nucleic acid signals were distributed specifically in the primary spermatocytes. Another DNA fragment derived from 1 week old rat testis, named AA11, was hybridized specifically in Sertoli cell of 1 week old rat testis. Northern blot hybridization with [α 32 P] dCTP labeled CG14 probe, including cardiac, liver, kidney, brain, testis, and epididymis tissue mRNAs of rat, showed that an mRNA specific hybridization band, size of 1.258 kb, was found in testis tissue and size of 1.531 kb of another hybridization band present in epididymis tissue. The CG14 DNA specific gene fragment existed in the λ ZAP II testis gene library. Conclusion 1. The DDRT PCR technique is a convenient tool to find the specific expression gene during spermatogenesis. 2. The CG14 DNA fragment was expressed significantly in rat testicular tissue, weakly expressed in the epididymis tissue of rat, but not found in cardiac, liver, kidney, and brain tissues. 3. The CG14 DNA fragment was specifically expressed in the primary spermatocytes and might be associated with the meiosis of the primary spermatocyte during spermatogenesis of rat. 4. The CG14 DNA fragment existed in the λ ZAP II testis gene library.
文摘Objective To investigate the role of fibronectin(FN) receptor α 5β 1 in liver fibrosis Methods Northern blot analysis and immuno^histochemical techniques were used to observe changes in the expression of FN and FN receptor α 5β 1 on hepatic stellate cells (HSCs) in vivo and in vitro of rat liver fibrosis induced by CCl 4 Results (1) α 5β 1 was mainly detected in the endothelia and some of the desmin(DM) positive cells of the sinusoids in normal rat liver The expression of α 5β 1 of DM positive cells detected by immunohistochemistry reached its peak at the 10th week of the experiment The changes in FN expression were similar to that of α 5β 1 (2) The expression of FN, α 5 and β 1 mRNAs in the experimental group was remarkably increased especially at the 6th week, compared to that of normal liver specimens The expression of the three mRNAs of HSCs in vitro isolated from the experimental group (6 weeks) was higher than those from normal liver (3) The expression of FN, α 5 and β 1 mRNAs increased in normal rat HSCs after treatment with transforming growth factor β 1 (TGF β 1) for 2 hours After 4 hours of treatment, the expression of the three mRNAs fell to levels similar to the control group Immunocytochemistry revealed that the expression of α 5β 1 of HSCs reached its peak after 4 hours of treatment with TGF β 1 and dropped to normal 2 hours later Conclusion These data suggest that HSCs normally express FN receptor α 5β 1 The activation of HSCs during liver fibrogenesis leads to an increase of FN, α 5 and β 1 mRNA expression The expression of FN and α 5β 1 of HSCs in vitro is up regulated by TFG α 5β 1 The detection of gene transcription of FN and its receptor by Northern blot analysis suggests the activation and proliferation of HSCs and thereby provides a sensitive signal of liver fibrosis
