睾丸间质细胞——研究自体吞噬的一种正常细胞模型

细胞在生理状态下自体吞噬出现的频率很低,很难用正常细胞来研究自体吞噬活动,一般都通过诱导自体吞噬来获得有关自体吞噬活动的资料。本实验观察了肝、肾、睾丸等组织的32种细胞,发现睾丸间质细胞中自体吞噬出现频率远远高于其他细胞,平均每100个细胞切面中可以看到25个自噬小体,从而为研究自体吞噬的过程和机理提供了一个正常细胞模型。本实验还观察到睾丸间质细胞的自体吞噬活动可分为前自噬小体、早期自噬小体和晚期自噬小体三个阶段,是一个连续的过程。前自噬小体和早期自噬小体不含溶酶体酶,只有在自噬小体与溶酶体接触后,才从后者获取溶酶体酶并将其内容物消化分解,成为晚期自噬小体。由自体吞噬所产生的残余体并不在睾丸间质细胞内积聚,而是通过胞吐作用排出细胞外。     

溶酶体的自体吞噬作用

溶酶体普遍存在于各种真核细胞中,它除了能够对进入细胞的有害异物起“消化”作用外,还能对细胞内由于生理或病理原因破坏或衰老的细胞器,甚至生物大分子起“消化”作用,以充份利用有用物质,清除细胞内废物,保障调节代谢的正常进行,这种作用称为自体吞噬作用。溶酶体的自体吞噬作用过程与异体吞噬作用过程基本相同,差别只是被消化物质不是细胞外来异物,而是细胞内源性物质。如衰老、破损的线粒体、内质网等细胞器及某些大分     

内分泌细胞中溶酶体对激素分泌调节的作用

本实验用外源性雄激素引起垂体促性腺激素细胞和睾丸间质细胞分泌抑制,对这两种细胞中的溶酶体及分泌吞噬和自体吞噬活动进行了超微结构形态观察和半定量分析。实验中应用了CMP酶细胞化学技术和免疫胶体金技术。研究结果显示,在分泌受抑制状态下,垂体促性腺激素细胞中溶酶体增多,分泌吞噬活动加强;与此同时,睾丸间质细胞也表现溶酶体增多、自体吞噬活动加强。这些结果不仅再次证明在分泌蛋白质激素细胞中溶酶体以分泌吞噬的方式参与了激素分泌调节,更重要的是初步证明在分泌类固醇激素细胞的分泌调节中,也有溶酶体的参与,其形式是自体吞噬作用。细胞通过自体吞噬作用得以在短时间内清除一部分合成激素的细胞器和其中的激素,这可能是分泌类固醇激素的细胞及时有效地调整激素分泌量的一项重要机制,与分泌蛋白质激素细胞的分泌吞噬有着相同的意义。     

多泡体形成过程的细胞化学研究

Multivesicular bodies were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testes. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase (CMPase) activity and in animals sacrificed at various time intervals ranging from 5 min to 2 hrs after a single intratesticular injection of cationic ferritin (CF). Analysis of morphological and cytochemical data led to the following interpretation for the origin and fate of the multivesicular bodies in Leydig cells. The formation of multivesicular bodies in Leydig cells can be divided into three steps. Step 1, some endocytic vacuoles in Golgi region fuse with small vesicles to form pre-multivesicular bodies. Step 2, the pre-multivesicular bodies fuse together to form pale multivesicular bodies which are characterized by their large size, pale matrix and paucity of internal vesicles. Step 3, the pale multivesicular bodies remove their surplus enveloping membrane to become dense multivesicular bodies which are characterized by their smaller size, dense matrix and filling with internal vesicles. The pre-multivesicular bodies and pale multivesicular bodies do not contain hydrolytic enzymes, the dense multivesicular bodies acquire their hydrolytic enzymes by fusion with lysosomes and show CMPase activity. The dense multivesicular bodies often show a very close association with autophagosomes, and they might be involved in the autophagic activity of Leydig cells.     

大鼠睾丸间质细胞的自体吞噬活动

本文结合超微结构和细胞化学观察,研究大鼠睾丸间质细胞(Leydig细胞)中溶酶体的结??构与功能。观察结果表明,大鼠睾丸间质细胞中高尔基体非常发达,在高尔基体的成熟面存在着CMP酶阳性反应的GERL系统,说明这种细胞有不断产生溶酶体的能力。细胞化学结果也证实在睾丸间质细胞有较多的初级和次级溶酶体。睾丸间质细胞不仅有较多的溶酶体,而且还有相当数量的自噬小体,存在着活跃的自体吞噬活动。自噬小体的界膜来源于特化的光面内质网或高尔基体膜囊,包围的内容物主要是光面内质网和少量线粒体。当自噬小体与溶酶体融合后即成为自体吞噬泡,由于酶的消化作用,自体吞噬泡内的细胞器有一系列形态变化。根据CMP酶细胞化学反应,可以区分自噬小体和自体吞噬泡,后者是一种次级溶酶体,呈CMP酶阳性反应。睾丸间质细胞是分泌雄性激素的内分泌细胞,其光面内质网和线粒体在类固醇激素分泌中起重要作用,自体吞噬活动的结果是去除部分内质网和线粒体,可能在细胞水平上起着对雄性激素分泌的调节作用。     

多泡体形成过程的细胞化学研究

本实验用酶细胞化学和示踪细胞化学方法观察了睾丸间质细胞中多泡体的形成过程及其与溶酶体的关系。实验结果表明,睾丸间质细胞中多泡体的形成可分三个阶段:首先,一些含内吞物质的泡状结构进入高尔基体区域,与那里的小泡融合,形成内含少量小泡的前多泡体;然后,前多泡体互相融合,形成体积较大、基质电子密度低、内含小泡排列稀疏的低电子密度多泡体;最后,低电子密度多泡体通过表面长出微绒毛样结构并不断断裂的方式去除多余的界膜,形成体积较小、基质电子密度高、内含小泡排列紧密的高电子密度多泡体。因此,多泡体的形成既与内吞活动有关,又与高尔基体区域小泡有关。前多泡体和低电子密度多泡体不含溶酶体酶。在多泡体形成过程中,只有到高电子密度多泡体阶段,才与溶酶体发生关系,从溶酶体中获取溶酶体酶。多泡体形成后,常与自体吞噬泡靠近,可能参与睾丸间质细胞的自体吞噬活动。     

逆向溶血斑法检测小鼠睾丸间质细胞睾酮的分泌

用逆向溶血斑法检测单个小鼠睾丸间质细胞睾酮的分泌,为进一步研究单个睾丸间质细胞的结构和功能提供一种有效的途径,结果表明,分泌睾酮的睾丸间质细胞周围形成空斑,空斑面积随睾酮分泌增加而增大,说明只要获得抗体,逆向溶血斑法就可以检测任何细胞的分泌物。     

不同移植部位对成年大鼠睾丸间质细胞雄激素分泌功能的影响

目的:探索不同移植部位对移植的成年SD大鼠睾丸中睾丸间质细胞存活及雄激素分泌功能的影响。方法:将健康成年雄性SD大鼠随机分为对照组、假手术组、皮下组和肾包膜组。对照组大鼠不去势,其余大鼠于睾丸移植前1周行去势手术。对照组和假手术组去势后仅行背部皮肤切开,不进行睾丸移植;皮下组背部两侧各移植1/3个成年SD大鼠睾丸组织;肾包膜组每侧肾包膜下移植1/3个成年SD大鼠睾丸组织。4周后取材行HE和免疫组化染色,分析移植睾丸组织中睾丸间质细胞存活情况,ELISA法检测受体大鼠血清睾酮水平。结果:皮下组和肾包膜组移植物中难于见到完整的睾丸间质组织,但免疫组化染色发现大量HSD-17β1阳性细胞,对照组、皮下组和肾包膜组的HSD-17β1阳性细胞数分别为(24.33±4.30)、(9.83±4.05)和(12.67±2.81)个,对照组与皮下组相比差异具有统计学意义(p0.05);ELISA分析发现对照组、假手术组、皮下组和肾包膜组的血清睾酮浓度分别为(3.81±1.32)、(0.28±0.08)、(0.44±0.13)和(0.90±0.31)ng/m L,肾包膜组血清睾酮浓度高于假手术组(p0.01)和皮下组(p0.05),而皮下组血清睾酮水平高于假手术组,但两者差异无统计学意义(p0.05)。结论:移植的成年大鼠睾丸组织中的睾丸间质细胞可在受体肾包膜下或皮下存活,但肾包膜下移植可能更加有利于睾丸间质细胞存活和雄激素分泌。     

氟对体外培养的小鼠睾丸间质细胞增殖和细胞凋亡的影响

目的 观察不同浓度氟化钠对睾丸间质细胞增殖和细胞凋亡的影响,为氟中毒的机制研究提供依据.方法 取体外培养的睾丸间质细胞,胰酶消化后制成单细胞悬液,常规培养,待细胞融合率达80％,且未出现细胞分化时,将细胞分4组,加入不同浓度的氟化钠染毒(0,5,10,20 mg/L)睾丸间质细胞,分别干预0,24,48,72,96,1...     

Leydig cell development

This review is about the study of the testis Leydig cells formation and development in prenatal and postnatal periods. Leydig cells of testis are the main place of synthesis and secretion of androgens including testosterone--the main male sexual hormone. Testosterone plays an important role in male reproduction regulation. There are two types (two populations) of Leydig cells during ontogenesis. The first type is fetal Leydig cells, which appear and function in the prenatal masculinization period of the male urogenital system. Another type is adult Leydig cells, which originate during sexual maturation postnatally. Fetal and adult Leydig cells pass the same stages both in the prenatal and postnatal periods. They are Leydig cell progenitors, immature Leydig cells and adult Leydig cells.     

A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats

Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.     

Subversion of cellular autophagy by Anaplasma phagocytophilum

Anaplasma phagocytophilum , the causative agent of human granulocytic anaplasmosis, is an obligatory intracellular pathogen. After entry into host cells, the bacterium is diverted from the endosomal pathway and replicates in a membrane-bound compartment devoid of endosomal or lysosomal markers. Here, we show that several hallmarks of early autophagosomes can be identified in A. phagocytophilum replicative inclusions, including a double-lipid bilayer membrane and colocalization with GFP-tagged LC3 and Beclin 1, the human homologues of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6 respectively. While the membrane-associated form of LC3, LC3-II, increased during A. phagocytophilum infection, A. phagocytophilum -containing inclusions enveloped with punctate GFP-LC3 did not colocalize with a lysosomal marker. Stimulation of autophagy by rapamycin favoured A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine did not inhibit A. phagocytophilum internalization, but reversibly arrested its growth. Although autophagy is considered part of the innate immune system that clears a variety of intracellular pathogens, our study implies that A. phagocytophilum subverts this system to establish itself in an early autophagosome-like compartment segregated from lysosomes to facilitate its proliferation.     

Inhibitive effects of Photofrin on cellular autophagy

Depending on the circumstances, autophagy can be either a protective or damaging cellular process. The role of autophagy in photodynamic therapy (PDT), a photo‐chemotherapy that utilizes light to activate a photosensitizer drug to achieve localized cellular damage, has been explored in recent years. It has been reported that autophagy in PDT is significantly influenced by the treatment protocol. In this work, the role of Photofrin, a well‐established clinical photosensitizer, in regulating cellular autophagy was investigated. The effects of Photofrin on cellular autophagy induced by conventional starvation or rapamycin techniques were studied. By fluorescence imaging, Western blotting and cell viability assays, it was found that Photofrin can effectively inhibit cellular autophagy induced by starvation or rapamycin. This autophagy blocking is independent of the photosensitizing property of the drug. With Baf‐A1, a well‐established agent that inhibits autophagosome from fusing with lysosome, we also found that, the observed phenomenon is not due to accelerated degradation of existing autophagosomes, thus proving that the drug Photofrin alone, without light excitation, can truly block autophagy. J. Cell. Physiol. 224: 414–422, 2010. © 2010 Wiley‐Liss, Inc.     

Antibody-dependent cellular cytotoxicity effector cell capability among normal individuals.

Nonadherent peripheral blood lymphocytes (PBL) from 110 normal individuals were evaluated for their capability to mediate antibody-dependent cellular cytotoxicity (ADCC) against rabbit antibody-sensitized chicken erythrocytes (CRBC). Titration of PBL against a constant number of chromium-51 labeled CRBC allowed for construction of dose response cytotoxicity curves where percent chromium release was plotted against the number of PBL. From these curves the maximum degree of lymphocyte-mediated cytotoxicity and the number of lymphoid cells required for 50% of the maximum lymphocyte-mediated cytotoxicity were determined. The results suggested that in this system the cytotoxic capability of PBL from normal individuals was independent of the age and sex of the donor. The cytotoxic capability of PBL was also found to be constant in individuals tested sequentially. However, significant differences were found to exist between different individuals in their capability of mediating ADCC in this system.     

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.