目的:探讨尿源干细胞(USC)对海绵体神经损伤性勃起功能障碍(CNIED)大鼠勃起功能和阴茎海绵体组织结构的保护作用。方法:60只成年雄性SD大鼠随机平均分为4组(n=15只/组):假手术组、双侧海绵体神经钳夹损伤组(BCNI组)、PBS组、USC组。假...目的:探讨尿源干细胞(USC)对海绵体神经损伤性勃起功能障碍(CNIED)大鼠勃起功能和阴茎海绵体组织结构的保护作用。方法:60只成年雄性SD大鼠随机平均分为4组(n=15只/组):假手术组、双侧海绵体神经钳夹损伤组(BCNI组)、PBS组、USC组。假手术组予暴露双侧海绵体神经后直接关闭手术切口,其余3组均予血管钳钳夹双侧海绵体神经1 min,建立CNIED模型;PBS组和USC组分别予阴茎海绵体注射PBS(200μl)或USC(1×10~6细胞/200μl PBS)。治疗28 d后测定大鼠的最大海绵体内压(mICP)和m ICP/平均动脉压(mICP/MAP),并通过Western印迹检测海绵窦内皮细胞标志物e NOS,平滑肌标志物α-SMA,以及Collagen I,通过免疫组化检测海绵体阴茎背神经内的神经标志物(nNOS、NF-200),Masson染色检测海绵体平滑肌/胶原比值,以及TUNEL染色检测海绵窦内细胞凋亡水平。结果:治疗28 d后,USC组大鼠的mICP及mICP/MAP均较PBS组[(81±9.9)mm Hg vs(31±8.3)mm Hg,0.72±0.05 vs 0.36±0.03,P<0.05]和BCNI组[(81±9.9)mm Hg vs(33±4.2)mm Hg,0.72±0.05 vs 0.35±0.04,P<0.05]显著升高。免疫组化结果显示:USC组大鼠背神经内n NOS、NF-200阳性神经纤维面积的比例(%)均较PBS组(11.31±4.22 vs 6.86±3.08,27.31±3.12 vs 17.38±2.87,P<0.05)和BCNI组(11.31±4.22 vs 7.29±4.84,27.31±3.12 vs 19.49±4.92,P<0.05)显著增加;Western印迹检测结果显示:USC组大鼠e NOS/GAPDH比值较PBS组(0.52±0.08 vs 0.31±0.06,P<0.05)和BCNI组(0.52±0.08 vs 0.33±0.07,P<0.05)均显著提高,α-SMA含量亦较PBS组(1.01±0.09 vs 0.36±0.05,P<0.05)和BCNI组(1.01±0.09 vs 0.38±0.04,P<0.05)显著提高,而Collagen I含量较PBS组(0.28±0.06 vs 0.68±0.04,P<0.05)和BCNI组(0.28±0.06 vs 0.70±0.10,P<0.05)显著降低;且Masson染色结果示USC组大鼠阴茎平滑肌/胶原比值(%)亦较PBS组(17.91±2.86 vs 7.70±3.12,P<0.05)和BCNI组(17.91±2.86 vs 8.21±3.83,P<0.05)显著升高。TUNEL染色显示USC组大鼠海绵窦内细胞的凋亡指数(%)较PBS组(3.31±0.83 vs 9.82±0.76,P<0.01)和BCNI组(3.31±0.83 vs 9.75±0.91,P<0.05)显著降低。结论:USC可以通过保护神经、改善海绵窦内皮功能和海绵体纤维化,抑制细胞凋亡,显著保护CNIED大鼠的勃起功能。展开更多
目的:探讨尿源干细胞来源的外泌体(USC-Exo)对糖尿病性勃起功能障碍(DED)大鼠海绵窦内皮功能和勃起功能的改善作用,并探讨其作用机制。方法:通过超速离心法从USC的培养基中提取USC-Exo,并进行鉴定。将SD大鼠海绵窦内皮细胞(CCEC)分为4组...目的:探讨尿源干细胞来源的外泌体(USC-Exo)对糖尿病性勃起功能障碍(DED)大鼠海绵窦内皮功能和勃起功能的改善作用,并探讨其作用机制。方法:通过超速离心法从USC的培养基中提取USC-Exo,并进行鉴定。将SD大鼠海绵窦内皮细胞(CCEC)分为4组:(1)正常组;(2)高糖组;(3)Exo组:高糖培养基中加入USC-Exo(10μg/ml);(4)3-MA组:高糖培养基中加入USC-Exo(10μg/ml)和3-MA(2 mmol/L)。将mRFP-GFP-LC3腺病毒转染CCEC后在荧光显微镜下检测细胞内自噬流的改变;通过CCK8实验检测细胞增殖能力的改变,通过Matrigel实验检测CCEC的成管能力。通过腹腔注射链脲佐菌素及阿扑吗啡实验筛选出DED大鼠,将其随机平均分为2组(n=5只/组):DED组和Exo组,另取5只同龄正常雄性大鼠作为正常组,其中正常组和DED组予阴茎海绵窦注射100μl PBS,Exo组予阴茎海绵体注射USC-Exo 100μl(浓度为1μg/μl)。治疗4周后测定大鼠最大海绵体内压(ICPmax)和平均动脉压(MAP),通过免疫荧光和Western印迹检测海绵窦内皮细胞标志物CD31,通过Western印迹检测自噬标志物Beclin1和LC3-I/II的蛋白表达,通过透射电镜检测海绵窦内皮细胞内自噬体的数量。结果:自噬流检测结果表明USC-Exo可以显著增加高糖培养CCEC内自噬体的数量(39.5±6.2 vs 12.5±5.4,P<0.05)。Western印迹结果显示Exo组CCEC的Beclin1蛋白表达水平显著高于高糖组(P<0.05)。CCK8结果显示Exo组CCEC的增殖能力较高糖组显著升高(P<0.05),而自噬抑制剂3-MA可以逆转USC-Exo对CCEC增殖能力的提升作用。Matrigel成管实验表明Exo组CCEC的成管能力较高糖组的显著提高(15.3±3.2 vs 6.3±2.1,P<0.05),同样加入3-MA后同样可以逆转这一作用。海绵体测压结果显示Exo组大鼠的ICPmax和ICPmax/MAP水平均较DED组显著升高[(86.6±12.6)mmHg vs(37.9±10.9)mmHg,89.3±14.1 vs 41.7±11.5,P<0.05]。Western印迹结果显示Exo组大鼠的海绵体内CD31、Beclin1和LC3-I/II的蛋白含量显著高于DED组大鼠(P<0.05)。透射电镜结果表明,Exo组大鼠阴茎海绵窦内皮细胞的自噬体数量显著多于DED组(3.7±0.6 vs 1.0±1.0,P<0.05)。结论:USC-Exo可以通过提高大鼠海绵窦内皮细胞的自噬水平,显著改善DED大鼠的海绵窦内皮功能和勃起功能。展开更多
To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines we...To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines were crossed with a highly susceptible variety LTH, and their F1 and F2 populations were inoculated by two representative blast isolates with broad pathogenicity to analyze their resistance inheritance basis. Meanwhile three mutant lines and the ground control were analyzed by 225 rice SSR （simple sequence repeat） primer pairs selected throughout the 12 chromosomes of whole rice genome, to scan the mutagenesis in genome of the mutant lines. The results indicated the blast-resistant genes harbored in these mutant lines were dominant. It was demonstrated that the resistance of mutant H1 to isolate GD0193 and GD3286 was controlled by a single gene, respectively; while mutants H2 and H3 were controlled by two pairs of major genes against isolate GD3286 and H2 showed complicated genetic mechanism to isolate GD0193. H3＇s resistance to isolate GD0193 was verified to be controlled by a single gene. According to the results of SSR analysis, three mutant lines showed different mutant rates as compared with the ground control, and the mutant rates also varied. Resistance genes can be induced from rice by space mutation, and different genomic variations were detected in blast-resistant lines.展开更多
Blast resistance and grain quality are major problems in hybrid rice production in China. In this study, two resistance （R） genes, Pi46 and Pita, along with the gene Wxb, which mainly affects rice endosperm amylose ...Blast resistance and grain quality are major problems in hybrid rice production in China. In this study, two resistance （R） genes, Pi46 and Pita, along with the gene Wxb, which mainly affects rice endosperm amylose content （AC）, were introgressed into an elite indica restoring line, R8166, which has little blast resistance and poor grain quality through marker-assisted selection （MAS）. Eight improved lines were found to have recurrent genome recovery ratios ranging from 88.68 to 96.23%. Two improved lines, R163 and R167, were selected for subsequent studies. R167, which has the highest recovery ratio （96.23%）, showed no significant differences in multiple agronomic traits. In contrast, R163 with the lowest recovery ratio （88.68%） exhibited significant differences in heading date and yield per plant compared with the recurrent parent. At two developmental stages, R163 and R167 had greatly enhanced resistance to blast over the recurrent parent. Similar trends were also observed for agronomic traits and blast resistance in R163- and R167-derived hybrids when compared with the counterparts from R8166. In addition, R163, R167, and their derived hybrids significantly improved the grain quality traits, including amylose content （AC）, gel consistency （GC）, chalky grain rate （CGR）, and degree of endosperm chalkiness （DEC）. It confirmed the success of efficiently developing elite restoring lines using MAS in this study.展开更多
Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed int...Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179（HH179） using the marker-assisted backcross breeding（MABB） procedure. As a result, three improved lines（e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita） were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines（NILs）. The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179（23.5%）. Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.展开更多
文摘目的:探讨尿源干细胞(USC)对海绵体神经损伤性勃起功能障碍(CNIED)大鼠勃起功能和阴茎海绵体组织结构的保护作用。方法:60只成年雄性SD大鼠随机平均分为4组(n=15只/组):假手术组、双侧海绵体神经钳夹损伤组(BCNI组)、PBS组、USC组。假手术组予暴露双侧海绵体神经后直接关闭手术切口,其余3组均予血管钳钳夹双侧海绵体神经1 min,建立CNIED模型;PBS组和USC组分别予阴茎海绵体注射PBS(200μl)或USC(1×10~6细胞/200μl PBS)。治疗28 d后测定大鼠的最大海绵体内压(mICP)和m ICP/平均动脉压(mICP/MAP),并通过Western印迹检测海绵窦内皮细胞标志物e NOS,平滑肌标志物α-SMA,以及Collagen I,通过免疫组化检测海绵体阴茎背神经内的神经标志物(nNOS、NF-200),Masson染色检测海绵体平滑肌/胶原比值,以及TUNEL染色检测海绵窦内细胞凋亡水平。结果:治疗28 d后,USC组大鼠的mICP及mICP/MAP均较PBS组[(81±9.9)mm Hg vs(31±8.3)mm Hg,0.72±0.05 vs 0.36±0.03,P<0.05]和BCNI组[(81±9.9)mm Hg vs(33±4.2)mm Hg,0.72±0.05 vs 0.35±0.04,P<0.05]显著升高。免疫组化结果显示:USC组大鼠背神经内n NOS、NF-200阳性神经纤维面积的比例(%)均较PBS组(11.31±4.22 vs 6.86±3.08,27.31±3.12 vs 17.38±2.87,P<0.05)和BCNI组(11.31±4.22 vs 7.29±4.84,27.31±3.12 vs 19.49±4.92,P<0.05)显著增加;Western印迹检测结果显示:USC组大鼠e NOS/GAPDH比值较PBS组(0.52±0.08 vs 0.31±0.06,P<0.05)和BCNI组(0.52±0.08 vs 0.33±0.07,P<0.05)均显著提高,α-SMA含量亦较PBS组(1.01±0.09 vs 0.36±0.05,P<0.05)和BCNI组(1.01±0.09 vs 0.38±0.04,P<0.05)显著提高,而Collagen I含量较PBS组(0.28±0.06 vs 0.68±0.04,P<0.05)和BCNI组(0.28±0.06 vs 0.70±0.10,P<0.05)显著降低;且Masson染色结果示USC组大鼠阴茎平滑肌/胶原比值(%)亦较PBS组(17.91±2.86 vs 7.70±3.12,P<0.05)和BCNI组(17.91±2.86 vs 8.21±3.83,P<0.05)显著升高。TUNEL染色显示USC组大鼠海绵窦内细胞的凋亡指数(%)较PBS组(3.31±0.83 vs 9.82±0.76,P<0.01)和BCNI组(3.31±0.83 vs 9.75±0.91,P<0.05)显著降低。结论:USC可以通过保护神经、改善海绵窦内皮功能和海绵体纤维化,抑制细胞凋亡,显著保护CNIED大鼠的勃起功能。
文摘目的:探讨尿源干细胞来源的外泌体(USC-Exo)对糖尿病性勃起功能障碍(DED)大鼠海绵窦内皮功能和勃起功能的改善作用,并探讨其作用机制。方法:通过超速离心法从USC的培养基中提取USC-Exo,并进行鉴定。将SD大鼠海绵窦内皮细胞(CCEC)分为4组:(1)正常组;(2)高糖组;(3)Exo组:高糖培养基中加入USC-Exo(10μg/ml);(4)3-MA组:高糖培养基中加入USC-Exo(10μg/ml)和3-MA(2 mmol/L)。将mRFP-GFP-LC3腺病毒转染CCEC后在荧光显微镜下检测细胞内自噬流的改变;通过CCK8实验检测细胞增殖能力的改变,通过Matrigel实验检测CCEC的成管能力。通过腹腔注射链脲佐菌素及阿扑吗啡实验筛选出DED大鼠,将其随机平均分为2组(n=5只/组):DED组和Exo组,另取5只同龄正常雄性大鼠作为正常组,其中正常组和DED组予阴茎海绵窦注射100μl PBS,Exo组予阴茎海绵体注射USC-Exo 100μl(浓度为1μg/μl)。治疗4周后测定大鼠最大海绵体内压(ICPmax)和平均动脉压(MAP),通过免疫荧光和Western印迹检测海绵窦内皮细胞标志物CD31,通过Western印迹检测自噬标志物Beclin1和LC3-I/II的蛋白表达,通过透射电镜检测海绵窦内皮细胞内自噬体的数量。结果:自噬流检测结果表明USC-Exo可以显著增加高糖培养CCEC内自噬体的数量(39.5±6.2 vs 12.5±5.4,P<0.05)。Western印迹结果显示Exo组CCEC的Beclin1蛋白表达水平显著高于高糖组(P<0.05)。CCK8结果显示Exo组CCEC的增殖能力较高糖组显著升高(P<0.05),而自噬抑制剂3-MA可以逆转USC-Exo对CCEC增殖能力的提升作用。Matrigel成管实验表明Exo组CCEC的成管能力较高糖组的显著提高(15.3±3.2 vs 6.3±2.1,P<0.05),同样加入3-MA后同样可以逆转这一作用。海绵体测压结果显示Exo组大鼠的ICPmax和ICPmax/MAP水平均较DED组显著升高[(86.6±12.6)mmHg vs(37.9±10.9)mmHg,89.3±14.1 vs 41.7±11.5,P<0.05]。Western印迹结果显示Exo组大鼠的海绵体内CD31、Beclin1和LC3-I/II的蛋白含量显著高于DED组大鼠(P<0.05)。透射电镜结果表明,Exo组大鼠阴茎海绵窦内皮细胞的自噬体数量显著多于DED组(3.7±0.6 vs 1.0±1.0,P<0.05)。结论:USC-Exo可以通过提高大鼠海绵窦内皮细胞的自噬水平,显著改善DED大鼠的海绵窦内皮功能和勃起功能。
基金supported by the National 863 Program of China (2002AA2410111)the Natural Science Foundation of Guangdong Province,China (031994)the Special Project of Key Science and Technology of Guangdong Province,China (2004A20107001)
文摘To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines were crossed with a highly susceptible variety LTH, and their F1 and F2 populations were inoculated by two representative blast isolates with broad pathogenicity to analyze their resistance inheritance basis. Meanwhile three mutant lines and the ground control were analyzed by 225 rice SSR （simple sequence repeat） primer pairs selected throughout the 12 chromosomes of whole rice genome, to scan the mutagenesis in genome of the mutant lines. The results indicated the blast-resistant genes harbored in these mutant lines were dominant. It was demonstrated that the resistance of mutant H1 to isolate GD0193 and GD3286 was controlled by a single gene, respectively; while mutants H2 and H3 were controlled by two pairs of major genes against isolate GD3286 and H2 showed complicated genetic mechanism to isolate GD0193. H3＇s resistance to isolate GD0193 was verified to be controlled by a single gene. According to the results of SSR analysis, three mutant lines showed different mutant rates as compared with the ground control, and the mutant rates also varied. Resistance genes can be induced from rice by space mutation, and different genomic variations were detected in blast-resistant lines.
基金supported by the grant from the State Scholarship Fund of China (20153069)the National Key R&D Program of China (2016YFD0101100)the earmarked fund for China Agriculture Research System (CARS-01-12)
文摘Blast resistance and grain quality are major problems in hybrid rice production in China. In this study, two resistance （R） genes, Pi46 and Pita, along with the gene Wxb, which mainly affects rice endosperm amylose content （AC）, were introgressed into an elite indica restoring line, R8166, which has little blast resistance and poor grain quality through marker-assisted selection （MAS）. Eight improved lines were found to have recurrent genome recovery ratios ranging from 88.68 to 96.23%. Two improved lines, R163 and R167, were selected for subsequent studies. R167, which has the highest recovery ratio （96.23%）, showed no significant differences in multiple agronomic traits. In contrast, R163 with the lowest recovery ratio （88.68%） exhibited significant differences in heading date and yield per plant compared with the recurrent parent. At two developmental stages, R163 and R167 had greatly enhanced resistance to blast over the recurrent parent. Similar trends were also observed for agronomic traits and blast resistance in R163- and R167-derived hybrids when compared with the counterparts from R8166. In addition, R163, R167, and their derived hybrids significantly improved the grain quality traits, including amylose content （AC）, gel consistency （GC）, chalky grain rate （CGR）, and degree of endosperm chalkiness （DEC）. It confirmed the success of efficiently developing elite restoring lines using MAS in this study.
基金supported by the grant from the Science and Technology Planning Project of Guangdong Province, China (2015A020209142)by the earmarked fund for the Modern Agro-industry Technology Research System,China (CARS-01-12)
文摘Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179（HH179） using the marker-assisted backcross breeding（MABB） procedure. As a result, three improved lines（e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita） were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines（NILs）. The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179（23.5%）. Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.
