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
Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to co...Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.展开更多
A patient referred to our hospital, diagnosed with left idiopathic chronic orchialgia, was evaluated with a thorough medical and psychiatric history, physical examination, scrotal ultrasound and magnetic resonance ima...A patient referred to our hospital, diagnosed with left idiopathic chronic orchialgia, was evaluated with a thorough medical and psychiatric history, physical examination, scrotal ultrasound and magnetic resonance imaging. Conservative management failed. The patient had temporary pain relief after undergoing outpatient cord block three times. Microsurgical denervation of the left spermatic cord was operated in March, 2011. A pain questionnaire was used to determine efficacy before and after operation, and complete pain relief was noted at one week after operation. The follow up period was 12 months, at the end of which the pain score was still zero. No complications, including testicular atrophy and hydrocele, occurred. Microsurgical denervation of the spermatic cord can be a minimally invasive, safe and effective management option for treatment of idiopathic chronic orchialgia.展开更多
Background Von HippeI-Lindau disease （VHL）,a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems,has rarely been reported in Asia.We genetically investigated a unique Chinese fa...Background Von HippeI-Lindau disease （VHL）,a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems,has rarely been reported in Asia.We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.Methods Genomic deoxyribonucleic acid （DNA） extracted from peripheral blood was amplified by polymerase chain reaction （PCR） to three exons of the VHL gene in 9 members of the Chinese family with VHL disease.PCR products were directly sequenced.We estimated the effects of VHL gene mutation on the stability of pVHL,which is indicated by the free energy difference between the wild-type and the mutant protein （△△G）.Results The Chinese family was classified as VHL type 1.Three family members,including two patients and a carrier,had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1.This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein.There was low stability of the VHL protein （the △△G was 12.71 kcal/mol） caused by this missense mutation.Conclusions We first reported a family with this VHL gene mutation in Asia.This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma （RCC） phenotype displayed by this family.The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.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。
文摘Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.
基金This study was supported by the grants from tlae National b]atural Science Foundation of China （No. 30901487, No. 81070488 and No. 81172432）, the Guangdong Natural Science Foundation （No. 9151802904000002）, and the Guangdong Provincial Ministry of Cooperation Project （No. 2011 B090400034）.
文摘A patient referred to our hospital, diagnosed with left idiopathic chronic orchialgia, was evaluated with a thorough medical and psychiatric history, physical examination, scrotal ultrasound and magnetic resonance imaging. Conservative management failed. The patient had temporary pain relief after undergoing outpatient cord block three times. Microsurgical denervation of the left spermatic cord was operated in March, 2011. A pain questionnaire was used to determine efficacy before and after operation, and complete pain relief was noted at one week after operation. The follow up period was 12 months, at the end of which the pain score was still zero. No complications, including testicular atrophy and hydrocele, occurred. Microsurgical denervation of the spermatic cord can be a minimally invasive, safe and effective management option for treatment of idiopathic chronic orchialgia.
基金This research was supported by grants from the National Natural Science Foundation of China （No. 30901487, No. 81302223, No. 81070488 and No. 81172432）, the Guangdong Natural Science Foundation （No. 10251008901000005）, and the Guangdong Province Science and Technology Project （No. 2011 B031800115, No. 2011 B032000003 and No. 20101051500032）.
文摘Background Von HippeI-Lindau disease （VHL）,a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems,has rarely been reported in Asia.We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.Methods Genomic deoxyribonucleic acid （DNA） extracted from peripheral blood was amplified by polymerase chain reaction （PCR） to three exons of the VHL gene in 9 members of the Chinese family with VHL disease.PCR products were directly sequenced.We estimated the effects of VHL gene mutation on the stability of pVHL,which is indicated by the free energy difference between the wild-type and the mutant protein （△△G）.Results The Chinese family was classified as VHL type 1.Three family members,including two patients and a carrier,had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1.This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein.There was low stability of the VHL protein （the △△G was 12.71 kcal/mol） caused by this missense mutation.Conclusions We first reported a family with this VHL gene mutation in Asia.This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma （RCC） phenotype displayed by this family.The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.
