大肠埃希菌感染诱导小鼠生精细胞凋亡的研究

目的探讨大肠埃希菌感染对雄性小鼠生精细胞凋亡以及对凋亡调控基因Fas/FasL的影响,进一步阐明大肠埃希菌感染致男性不育的分子机理。方法用大肠埃希菌直接注入成年雄性小鼠膀胱内模拟逆行感染的途径,感染后第7d处死小鼠,取睾丸组织分别作HE染色和用TUNEL法观察生精细胞凋亡及睾丸的病理变化;免疫组化法检测Fas/FasL在睾丸组织中的表达,同时检测精子数量、精子活动率和精子畸形率。结果大肠埃希菌感染小鼠后生精细胞的凋亡指数及Fas和FasL表达水平分别为(18.50±1.50)%、(48.10±4.19)%和(34.60±4.18)%,对照组分别为(7.40±1.69)%、(23.50±2.66)%和(19.10±4.30)%,差异均有统计学意义(P均<0.05)。感染组精子数量、精子活动率和精子畸形率分别为(6.00±1.41)×106/ml、(59.80±5.29)%和(14.78±2.03)%,对照组分别为(12.00±4.12)×106/ml、(88.90±5.37)%和(5.88±1.10)%,感染组和正常对照组比较差异均有统计学意义(P均<0.01)。感染组小鼠睾丸组织出现明显的病理变化。结论雄性小鼠感染大肠埃希菌后,可导致生精细胞凋亡增加及上调Fas和FasL蛋白的表达水平;Fas和FasL在小鼠睾丸组织的高表达诱导生精细胞的凋亡,最终使小鼠精子形成减少,活动率下降,畸形率增加。大肠埃希菌感染造成生精细胞凋亡可能是导致男性不育的原因之一。     

男性不育精浆弓形虫感染与生精细胞凋亡关系的研究

目的探讨男性不育精浆弓形虫感染与生精细胞凋亡及精子凋亡与男性不育关系。方法应用末端脱氧核苷酸转移酶(TdT)介导的脱氧核苷酸原位末端标记法(TUNEL)检测正常生育、不育男性精液中的生精细胞凋亡。结果男性不育精浆弓形虫感染组生精细胞凋亡率为(14．17± 7．16)％,显著高于男性不育弓形虫感染阴性组生精细胞凋亡率(12．22±6．18)％(P<0．05)。男性不育组精子凋亡发生率为(13．76±9．19)％,显著高于正常生育组男性精子凋亡发生率(4．28± 1．67)％(P<0．01)。结论精子凋亡与男性不育有着密切关系,弓形虫感染可引起生精细胞凋亡。     

弓形虫急性感染小鼠睾丸的病理学及发病机理的研究

目的　观察弓形虫急性感染小鼠睾丸的病理学变化及探讨其发病机理。方法　将弓形虫急性感染和正常对照NIH小鼠睾丸做印片及切片 ,观察生精细胞的病理变化及弓形虫侵入细胞的情况 ;应用免疫组化S -P法进行弓形虫抗原及bcl 2、c myc、P53、bax的检测。结果　弓形虫急性感染组小鼠睾丸印片中见生精细胞胞质及核内弓形虫速殖子 ;睾丸切片病理变化为生精停滞 ,精原细胞胞质空泡性变。免疫组化染色显示弓形虫 ;bcl 2及c myc在感染组和正常组间的表达无统计学意义 (P >0 .0 5 ) ;P53及bax在两组间均未表达。结论　弓形虫急性感染小鼠睾丸生精停滞等病理变化可能是弓形虫及其分泌的毒素直接损害、干扰生精细胞生成及分裂发生障碍和死亡的结果 ,与凋亡基因无明显关系     

弓形虫急性感染小鼠睾丸的病理学及发病机理的研究揪

目的观察弓形虫急性感染小鼠睾丸的病理学变化及探讨其发病机理.方法将弓形虫急性感染和正常对照NIH小鼠睾丸做印片及切片,观察生精细胞的病理变化及弓形虫侵入细胞的情况;应用免疫组化S-P法进行弓形虫抗原及bcl-2、c-myc、P53、bax的检测.结果弓形虫急性感染组小鼠睾丸印片中见生精细胞胞质及核内弓形虫速殖子;睾丸切片病理变化为生精停滞,精原细胞胞质空泡性变.免疫组化染色显示弓形虫;bcl-2及c-myc在感染组和正常组间的表达无统计学意义(P＞0.05);P53及bax在两组间均未表达.结论弓形虫急性感染小鼠睾丸生精停滞等病理变化可能是弓形虫及其分泌的毒素直接损害、干扰生精细胞生成及分裂发生障碍和死亡的结果,与凋亡基因无明显关系.     

弓形虫感染与生精细胞凋亡关系的研究

目的 探讨弓形虫感染与生精细胞凋亡的关系。方法 采用瑞-姬染色和末端脱氧核苷酸转移酶(TdT)介导的脱氧核苷酸原位末端标记法(TUNEL)检测弓形虫感染小鼠精子悬液的凋亡生精细胞。结果 弓形虫感染组生精细胞凋亡率明显高于正常对照组(P<0.05)。结论 弓形虫感染可能有促进生精细胞凋亡的作用。     

蒺藜皂苷对环磷酰胺致小鼠生精细胞凋亡的保护作用

目的研究蒺藜皂苷对环磷酰胺(cyclophosphamide,CP)所致小鼠生精细胞凋亡的保护作用,探讨蒺藜皂苷对生殖功能障碍小鼠的保护机制。方法将8周龄的雄性昆明种小鼠随机分为3组,对照组采取腹腔注射生理盐水,模型组和治疗组采取连续5d腹腔注射CP 80mg/(kg·d),腹腔注射同时将对照组和模型组小鼠连续灌胃生理盐水,治疗组小鼠连续灌胃蒺藜皂苷200mg/(kg·d)35d。比较各组小鼠体质比及睾丸重量;检测附睾精子密度、活精子百分率、精子活力和精子畸形率;HE染色观察睾丸生精上皮的发育;TUNEL法检测生精细胞的凋亡;Western blot检测睾丸组织中凋亡相关蛋白Caspase-8表达水平的变化。结果与模型组比较,蒺藜皂苷显著提高了生精功能损伤小鼠的精子密度、活精子百分比、精子活力,降低了精子畸形率,促进了生精小管上皮精原细胞及精子细胞的发育,上调了睾丸组织中Caspase-8的表达水平,使生精细胞的凋亡减少。结论蒺藜皂苷能显著修复CP所致的小鼠睾丸损伤,其修复机制可能通过上调Caspase-8的表达,拮抗环磷酰胺引起的生精功能损伤,减少生精细胞的凋亡。     

刚地弓形虫感染对小鼠生精细胞凋亡的影响

目的探讨刚地弓形虫感染对小鼠生精细胞(单倍体、二倍体、四倍体)凋亡的影响程度,进一步阐明弓形虫致雄性不育的机制。方法选用9~10周龄雄性BALB/c小鼠60只,随机分为PBS正常对照组、刚地弓形虫纯化速殖子感染组(2.5×103、5×103、1×104、2×104)及环磷酰胺阳性对照组。采用腹腔注射法建立刚地弓形虫感染致小鼠睾丸损伤的模型,病理切片观察,流式细胞仪检测各组小鼠睾丸各倍体生精细胞的凋亡情况,免疫组化测定睾丸凋亡蛋白bcl-2、bax的变化,进行图像分析。结果正常对照组未出现明显的凋亡峰,实验组小鼠睾丸的单倍体、二倍体细胞均出现明显凋亡,尤以二倍体明显,同时四倍体细胞明显减少;睾丸细胞bcl-2的表达无明显改变,但bax的表达量随攻虫剂量增加而增加。结论弓形虫可致小鼠睾丸各级生精细胞凋亡,对二倍体细胞的影响尤为明显。     

先天性睾丸发育不良43例临床分析

43例先天性睾丸发育不良患者中,无精36例（83.7%）,少精7例（16.3%）;染色体核型异常9例（32.1%）,染色体正常19例（67.9%）;睾丸活检示有生精细胞和精子发生者4例（26.7%）。认为先天性睾丸发育不良患者的细胞核型并非完全一致;睾丸组织病理改变波动于生精细胞完全缺如和有生精细胞及活跃的精子发生;主要治疗措施为长期乃至终生的雄激素补充治疗。     

痢疾杆菌D_(15)及其L型感染与细胞凋亡

目的　探讨痢疾杆菌及其L型感染与细胞凋亡的关系。方法　 6～ 8周龄C57BL/ 6J小鼠 ,随机分成 3组。A组 :痢疾杆菌D15感染组 ,每天每只经口灌注 0 5ml菌液 ,连续 12d。B组 :痢疾杆菌D15L型感染组 ,灌注时间及次数均同A组。C组 :生理盐水对照组。于实验第 13d处死动物 ,病变脏器超薄切片 ,用末端转移酶作原位缺口末端标记法检测各组小鼠主要脏器的凋亡细胞。结果　痢疾杆菌及其L型感染均可引起肠及肝细胞的凋亡 ,统计学处理显示 ,细菌型比L型更易引起细胞凋亡。结论　痢疾杆菌及其L型感染均可引起细胞凋亡     

清利生精丸对大肠埃希菌感染小鼠生殖细胞凋亡及P53蛋白表达的影响

目的探讨大肠埃希菌感染对雄性小鼠生殖细胞凋亡和P53蛋白表达的影响,从分子水平阐明清利生精丸治疗大肠埃希菌感染所致男性不育的机制。方法 50只雄性小鼠,经膀胱注射大肠埃希菌,建立动物感染模型,观察15d后随机分为5组:感染组(不治疗)、清利生精丸大、中、小等3种浓度治疗组(药物浓度分别为22.5、13.5和4.50g/ml)、呋喃坦啶治疗组,分别编码为MN、MTa、MTb、MTc、MTd组,另取10只小鼠膀胱注射生理盐水作为正常对照组(编码为CT)。各治疗组连续灌胃药液10d,应用流式细胞术检测小鼠睾丸生殖细胞凋亡率,免疫组化技术测定生殖细胞P53蛋白表达情况。结果各组小鼠均有一定的睾丸生殖细胞凋亡率,其中MN组生殖细胞凋亡率57.44%,CT组28.54%,MTa组30.11%,MTb组28.59%,差异有统计学意义(P<0.01);MN组生殖组细胞凋亡率与MTc(46.54%)和MTd组(43.41%)比较差异无统计学意义(P>0.05)。MN、CT、清利生精丸各治疗组和MTd组表达P53蛋白睾丸生殖细胞百分比差异有统计学意义(P<0.01)。结论大肠埃希菌感染小鼠后,可导致小鼠睾丸生殖细胞P53蛋白的表...     

Immunological,paracrine and endocrine aspects of testicular immune privilege

Protection of the spermatogenic cells from the host immune response is fundamental to male fertility. Significantly, this protection extends to the tolerance of foreign tissue grafts placed within the testicular environment, a phenomenon that is called ‘immune privilege’. This privilege of the testis appears to involve several levels of immune control, encompassing the normal mechanisms of immune tolerance, antigen sequestration behind the blood–testis barrier, reduced immune activation, localised immunosuppression and antigen-specific immunoregulation. Central to these regulatory processes are the somatic cells of the testis, particularly the Sertoli cells, and testicular secretions, including androgens, cytokines, peptides and bioactive lipids. Failure of these protective mechanisms, which may be precipitated by trauma, inflammation or infection, or as the consequence of genetic factors, can lead to androgen deficiency, infertility and autoimmunity.     

Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis

The studies described herein were designed to examine whether there is a threshold concentration of testosterone (T) within the seminiferous tubules that is required to maintain spermatogenesis in the rat, or alternatively, whether there is a dose-response relationship between the intratesticular T concentration and the maintenance of spermatogenesis. T was administered to intact adult male rats via sustained release polydimethylsiloxane capsules in order to experimentally clamp T at well defined concentrations within the seminiferous tubules. Implantation of T-filled capsules of increasing sizes resulted in linear increases in T concentrations in serum, interstitial fluid, and seminiferous tubule fluid (STF). We examined the effect of step decreases in intratesticular T concentration on the numbers of advanced spermatogenic cells maintained by the testis over a 2-month period. Quantitatively complete spermatogenesis was maintained despite an 80% reduction in the STF T concentration (to approximately 13 ng/ml) from control values. The ability of the testis to maintain complete spermatogenesis was extremely sensitive to further decreases in STF T concentration. Thus, reduction of the STF T concentration from approximately 13 to 9 ng/ml resulted in a reduction in the number of advanced spermatids that were maintained in the testis by approximately 100 x 10(6). Reduction of the STF T concentration to approximately 4 ng/ml resulted in a further reduction in the number of advanced spermatids per testis by 100 x 10(6). Taken together, these data support the contention that there is far more T present within the seminiferous tubules of intact rat testes than is required to maintain quantitatively normal spermatogenesis and reveal for the first time that there is a dose-response relationship between the STF T concentration and the quantitative maintenance of advanced spermatogenic cells in the rat testis.     

原位杂交检测喉癌中金葡菌L型DNA

目的探讨喉癌细胞中是否有金葡菌L型DNA原位杂交阳性表达。方法应用原位核酸杂交技术检测20例喉癌细胞核内金葡菌L型DNA的表达。结果60％癌细胞核、浆内显示金葡菌L型DNA阳性信号。免疫组化染色（S—P法）金葡菌L型抗原阳性率为75％。革兰氏染色金葡菌L型阳性率为70％。结论金葡菌L型DNA已进入喉癌细胞内,极有可能与宿主细胞DNA整合,从基因水平上影响细胞增生和癌变,提示金葡菌及其L型感染与喉癌发生和发展可能有密切的关系[6]。     

The Y chromosome-linked copy number variations and male fertility

Since the first definition of the AZoospermia Factor (AZF) regions, the Y chromosome has become an important target for studies aimed to identify genetic factors involved in male infertility. This chromosome is enriched with genes expressed exclusively or prevalently in the testis and their absence or reduction of their dosage is associated with spermatogenic impairment. Due to its peculiar structure, full of repeated homologous sequences, the Y chromosome is predisposed to structural rearrangements, especially deletions/ duplications. This review discusses what is currently known about clinically relevant Y chromosome structural variations in male fertility, mainly focusing on copy number variations (CNVs). These CNVs include classical AZF deletions, gr/gr deletion and TSPY1 CNV. AZF deletions are in a clear-cut causeeffect relationship with spermatogenic failure and they also have a prognostic value for testis biopsy. gr/gr deletion represents the unique example in andrology of a proven genetic risk factor, providing an eight-fold increased risk for oligozoospermia in the Italian population. Studies on TSPY1 CNV have opened new perspectives on the role of this gene in spermatogenic efficiency. Although studies on the Y chromosome have importantly contributed to the identification of new genetic causes and thus to the improvement of the diagnostic work-up for severe male factor infertility, there is still about 50% of infertile men in whom the etiology remains unknown. While searching for new genetic factors on other chromosomes, our work on the Y chromosome still needs to be completed, with special focus on the biological function of the Y genes.     

消化性溃疡与幽门螺杆菌L型感染相关性研究

目的　探讨消化性溃疡 (PU)与幽门螺杆菌 L型 (HP- L )感染的关系。方法　取 386例 PU患者胃窦、胃体及十二指肠粘膜组织 ,常规切片后以革兰氏染色和免疫组化染色镜检 HP- L 型细菌 ,并计算检出率。结果　 HP- L 型检出率为 5 3.37% ,其中革兰氏染色和免疫组化染色检出率分别为 5 6 .73%、5 4 .2 9% ,差异无显著性(P>0 .0 5 ) ;胃溃疡、十二指肠溃疡 HP- L 型检出率分别为 5 8.33%、5 5 .5 0 % ,差异无显著性 (P>0 .0 5 ) ;男女患者HP- L 检出率前者 (6 1.18% )明显高于后者 (38.17% ) (P<0 .0 1) ;30岁以下、30岁～、4 0岁～、5 0岁～患者 HP- L检出率依次为 32 .0 5 %、4 1.94 %、5 9.18%、71.79% ,差异显著 (P<0 .0 1)。结论　 PU患者 Hp- L型感染率较高 ,且男性高于女性 ,HP- L型检出率随年龄增长而增高。 PU患者 HP- L型变异可能是导致溃疡迁延不愈、反复发作的重要原因之一     

From the Cover: Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice

Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by the culture method without any genetic manipulations. The Sl/Sl^d mouse is used as a model of such male infertility. The testis of the Sl/Sl^d mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl^d mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.Spermatogenic impairments can be caused by malfunctions of either the germ cell itself or surrounding somatic cells, which collectively constitute the microenvironment for proper spermatogenesis. It is well known that the microenvironmental condition in the testis is under the body’s systemic control, particularly through hormones from the pituitary. It is also well recognized that testicular somatic cells, Sertoli cells in particular, play a pivotal role in spermatogenesis (1, 2). Although it remains to be elucidated what molecules are essential constituents of that microenvironment and how they exert their role in spatial and temporal terms, it is a reasonable assumption that the spermatogenic impairments caused by microenvironmental factors are treatable by correcting the defect(s).Recently, we succeeded in inducing complete spermatogenesis in mice using an organ culture method (3–5). In this culture system, germ cells developed from spermatogonial stem cells through mitotic differentiation of spermatogonia, meiosis in spermatocytes, and morphological transformation in haploid spermatids, to sperm. One of the greatest advantages of the in vitro system is that the culture medium can be modified freely to an extent that would be impossible to achieve in vivo. In the present study, we attempted to treat spermatogenic failure of a mutant mouse by culturing testis tissue fragments in media supplemented with combinations of growth factors.