Influence of age, hormones and germ cells on glutathione S-transferase activity in cultured Sertoli cells

Glutathione S-transferase (GSH-S-T) activity was measured, using 1-Cl-2,4-dinitrobenzene as substrate, in Sertoli cell cultures obtained from rats aged 10, 18, and 26 days. The GSH-S-T activity showed a significant increase with age of the Sertoli cell donor. When cultures were treated with hypotonic solution, in order to eliminate residual contaminating germ cells, the age dependent increase in enzyme activity was less pronounced. FSH, but not testosterone, increased enzyme activity in all cultures. Addition of freshly isolated germ cells (mainly pachytene spermatocytes) to hypotonic-treated Sertoli cell monolayers enhanced GSH-S-T activity at all ages. It is concluded that GSH-S-T activity can be measured in cultured Sertoli cells during the period of onset of spermatogenesis (10-26 days). This enzyme activity is dependent on age of the Sertoli cell donor and is influenced by FSH and germ cells. Since GSH-S-Ts are actively engaged in cell detoxificative functions through conjugation of xenobiotics with glutathione, the present findings suggest that this enzyme may have a relevant protective role during the critical period when spermatogenesis is being established.     

Possible role of arachidonic acid in the regulation of lactate production in rat Sertoli cells

The aim of the study was to determine whether arachidonic acid (AA) is involved in the regulation of Sertoli cell lactate production and if this fatty acid participates in follicle-stimulating hormone (FSH) regulation of Sertoli cell function. In a first set of experiments the effect of AA and porcine pancreas phospholipase A2 (PLA2) on lactate production, glucose uptake, lactate dehydrogenase (LDH) activity and LDH A mRNA levels in Sertoli cell cultures obtained from 20-day-old rats was evaluated. In a second set of experiments the effect of two PLA2 inhibitors--quinacrine (Q) and AACOCF3--on FSH stimulation of the above-mentioned parameters of Sertoli cell function was investigated. Treatment with PLA2 and AA increased Sertoli cell lactate production. The observed action of exogenously added PLA2 involved its catalytic properties responsible for AA release. PLA2 and AA treatments also stimulated Sertoli cell glucose uptake, LDH activity and LDH A mRNA levels. In order to determine whether AA participates in FSH regulation of Sertoli cell lactate production cells were incubated with FSH in the absence or presence of the PLA2 inhibitors Q and AACOCF3. Both drugs partially inhibited the ability of FSH to stimulate lactate production, glucose uptake and LDH activity. The present investigation suggests that AA is involved in the regulation of lactate production, glucose transport, LDH activity and LDH A mRNA levels. In addition, these results suggest that cytosolic PLA2 and AA may participate in FSH-regulation of Sertoli cell energetic metabolism.     

不同温度下体外大鼠睾丸支持细胞胶质细胞源性神经生长因子的表达

目的:通过观察不同温度下体外大鼠睾丸支持细胞(Sertoli cell,SC)胶质细胞源性神经生长因子(GDNF)表达特点,探讨高温致不育的机制。方法:采用组合酶消化法和选择性贴壁法分离雄性Wistar大鼠睾丸SC,将分离的SC分别置于不同温度进行体外培养,观察其贴壁、形态学变化,FasL免疫组化鉴定。实验分为对照组(35℃)、实验组(36℃、37℃、38℃、39℃)。CCK-8法检测SC增殖情况,HE染色观察细胞形态及结构,RTPCR、免疫荧光及Western印迹检测细胞GDNF的表达。结果:本实验条件下,体外分离培养SC纯度=(95.30±2.15)%(n=10),CCK-8实验结果显示,36℃时细胞增殖率最高(P0.01),36℃时,随着温度的提高,增殖率逐渐降低,39℃对细胞增殖具有明显抑制作用(P0.01);免疫荧光结果显示,GDNF表达于SC胞质,36℃时荧光最强,RT-PCR及Western印迹检测结果显示,高于36℃时,GDNF mRNA及蛋白的表达随着温度的增加而降低,36℃、37℃、38℃、39℃4组与对照组比较,差异均有统计学意义(P0.05)。结论:不同温度下,体外培养SC的增殖能力和GDNF表达明显不同。36℃时,随着温度的提高,增殖能力受到抑制,GDNF表达水平显著降低。本研究结果证实,高温下大鼠睾丸SC正常功能受到抑制,从而影响生精功能。     

不同浓度碱性成纤维细胞生长因子对体外培养兔肌腱细胞增殖的影响

目的探讨不同浓度的碱性成纤维细胞生长因子(bFGF)对体外培养兔肌腱细胞增殖的影响,并确定促进肌腱细胞增殖的最佳浓度。方法在体外培养第2代兔肌腱细胞的培养液中分别加入不同浓度(0.5、1、2、5、10、20、30、40、50μg/L)的bFGF,继续培养48 h,噻唑蓝(MTT)法染色,在酶联免疫检测仪上测出不同浓度bFGF组光密度(OD)值。结果与对照组OD均值相比:5、10、20、30、40、50μg/L bFGF组差异均有统计学意义(P<0.05);各组OD均值随bFGF浓度的增加,先逐渐增大(5～20μg/L),达到峰值后又逐渐降低(20～50μg/L)。结论 bFGF有明显促肌腱细胞增殖的作用,且与浓度有一定相关性,即促肌腱细胞增殖的起始bFGF浓度为5μg/L,而20μg/L时可达到促肌腱细胞增殖的最佳浓度。     

ADSC-bFGF对大鼠背部带蒂皮瓣存活率的实验研究

目的:脂肪来源间充质干细胞复合碱性成纤维细胞生长因子(ADSC-bFGF)对大鼠背部带蒂皮瓣模型存活率的影响。方法:获得SD大鼠腹股沟脂肪,经体外分离、培养并进行多功能分化潜能的鉴定。MTT法检测bFGF培养液对ADSC的影响。建立大鼠背部自身对照带蒂皮瓣模型。应用CM-DiI荧光染料标记ADSC。在皮瓣可预测坏死区域分别给予A1组bFGF、A2组ADSC、A3组bFGF-ADSC、B组生理盐水。全部动物于术后7天测量皮瓣成活面积、HE染色、冰冻切片的CD31荧光显影,统计学分析ADSC-bFGF促进皮瓣成活的作用。结果:bFGF有明显促ADSC增殖作用,增值最快的最低浓度为5μg/L。A3组皮瓣成活率、毛细血管增生均显著高于其它组。结论:bFGF可促ADSC增值,具有协同作用。ADSC-bFGF可高效的刺激大鼠背部自身对照带蒂皮瓣的血管新生,显著的提高皮瓣的成活率。     

Ontogenetic profile and thyroid hormone regulation of type-1 and type-8 glucose transporters in rat Sertoli cells

The glucose transporters (GLUTs) gene encode glycoproteins responsible for facilitating transfer of glucose across plasma membrane. In testis, different members of this family are present. In particular the main GLUT mRNA expression within the adult testis is the type 8, while type 1 is more expressed in prepubertal testis. Thyroid hormone, which receptors and function have been characterized in the testis, plays a crucial role in the cellular energetic metabolism. In fact, in the immature Sertoli cells, GLUT1 is up regulated by l-triiodothyronine (T(3)). The aim of this paper is to investigate the expression profile of GLUT1 and GLUT8 in the testis during development and in adulthood and analyse the role of T(3) on their expression. To analyse the expression of GLUT8 and GLUT1 we performed Northern blot and RT-PCR experiments in the whole testis and in Sertoli cells from rats of different ages. Treatments in vivo and in vitro with T(3) were used to study the effect of thyroid hormones on GLUT1 and GLUT8 expression. The activity of the rat GLUT1 promoter and its regulation by T(3) was studied with transient transfections in gonadal and non-gonadal cell lines and in primary Sertoli cell cultures. GLUT8 is expressed at a low level in the prepubertal testis and Sertoli cells and does not appear to be under T(3) control. GLUT1 is the predominant form in immature Sertoli cells. The effect of T(3) on its mRNA accumulation was quantified and confirmed by RT-PCR (control: 0.65 +/- 0.17; T(3): 1.23 +/- 0.04, arbitrary units, p < 0.05). However, transfection experiments showed that T(3) does not directly regulate GLUT1 promoter in any cell line tested. This is confirmed by the evidence that, upon extensive analysis, the rat GLUT1 promoter and the first intron sequence do not shows any thyroid responsive elements. Our data demonstrate that GLUT1 and GLUT8 are both expressed in prepubertal testis, but only GLUT1 is regulated by T(3). In addition, we found that the effect of T(3) cannot be attributed to its action on GLUT1 promoter.     

Effect of mirabegron on tight junction molecules in primary cultured rat Sertoli cells

Mirabegron is a selective beta3‐adrenoceptor (β₃‐AR) agonist, which is commonly used for the treatment of overactive bladder. This medicine is associated with atrophy of reproductive organs in rats. However, no study has examined the detailed action and mechanism of its toxicity in reproductive cells. In this study, we examined the effect of mirabegron on primary cultured rat Sertoli cells. Firstly, RT‐PCR and immunocytochemistry revealed that β₃‐AR was present in rat Sertoli cells. Then, primary cultured rat Sertoli cells were treated with mirabegron. Quantitative real‐time PCR revealed that mirabegron treatment induced a significant increase in claudin‐11 mRNA, which is crucial for spermatogenesis. Western blot analysis also showed that mirabegron treatment significantly activated p44/42 mitogen‐activated protein kinase (MAPK). After additional treatment with U0126, a specific noncompetitive inhibitor of mitogen‐activated protein kinase kinase (MAPKK), the upregulation of claudin‐11 mRNA induced by mirabegron was reduced. At the same time, immunocytochemistry showed mirabegron treatment disturbed claudin‐11 localisation to tight junction, which was recovered when treated with mirabegron in the presence of U0126. These results suggest that mirabegron treatment is associated with assembly of the blood–testis barrier through p44/42 MAPK pathway. These findings could explain one of the underlying mechanisms of reproductive toxicity induced by mirabegron.     

大鼠睾丸支持细胞溶酶体的周期性变化

作者在透射电镜下观察了大鼠精子发生过程中支持细胞（Ｓｅｒｔｏｌｉ细胞）中溶酶体的结构及分布的改变。结果Ｓｅｒｔｏｌｉ细胞中溶酶体数目多，在细胞中所处的位置有周期性变化，细胞中全部胞质和在基底部胞质中溶酶体的截面积占胞质截面积的比例在生精第ＶＩＩ阶段最大，近腔部溶酶体截面积占胞质截面积在第ＩＩ、ＩＸ阶段最小；Ｓｅｒｔｏｌｉ细胞总胞质和在基底部单位胞质截面积的溶酶体数在第ＶＩＩ阶段最大，近腔部单位胞质截面积溶酶体数在顶体期后几阶段最大，第ＩＸ阶段最小。Ｓｅｒｔｏｌｉ细胞溶酶体的周期性变化是与生精细胞相互影响、相互作用，也是精子连续正常发生的前提条件之一。     

Effect of insulin-like growth factor 1 and basic fibroblast growth factor on DNA synthesis and collagen production in cultured anterior cruciate ligament cells

This study was undertaken to investigate the effects of insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF) on the DNA and matrix synthesis of cells out-grown from the anterior cruciate ligament (ACL). Five batches of ACL cells from five 8-week-old Japanese white rabbits were isolated and maintained in culture until the fifth passage. We analyzed the effects of various concentrations of IGF-1 (1–1000 ng/ml) on [³H]-thymidine uptake in the cells at the first and fifth passages, and collagen content in the cell layer at the third passage, in the presence or absence of bFGF (10ng/ml). In the absence of bFGF, IGF-1 caused a significant increase in the synthesis of DNA and collagen in the ACL cells. IGF-1 and bFGF, in combination, synergistically increased the DNA synthesis of ACL cells, whereas such synergistic enhancement was not observed in their, collagen production. The amounts of [³H]-thymidine incorporated into the cells incubated with IGF-1 (500ng/ml) and bFGF (10ng/ml) combined were 1.3–1.5 times greater at first passage and 1.3–1.9 times greater at fifth passage than the sum of these with the growth factors used individually. Based on this in vitro finding, we consider it clinically relevant that IGF-1 and bFGF, when used together, have the capability to enhance the primary healing of ruptured ACL. Presented at the 11th Annual Meeting for Orthopaedic Research of the Japanese Orthopaedic Association, Kagoshima, Japan, October 17, 1996.     

Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice： role of human FSH receptor promoter

Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5“-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.     

bFGF和TGF-β1对原代培养的前列腺间质细胞的作用

目的:探讨碱性成纤维细胞生长因子(bFGF)和转化生长因子β1(TGF-β1)在良性前列腺增生(BPH)中的作用。方法:培养了人BPH间质细胞,采用MTT法检测无血清培养的间质细胞的增殖,用免疫组化方法检测平滑肌细胞表型变化,观察不同浓度bFGF和TGF-β1对培养的人BPH间质细胞的影响。结果:bFGF促进间质细胞增殖(P<0.05、P<0.01),较高浓度时(10μg/L)降低平滑肌细胞表型表达;TGF-β1(>0.1μg/L)抑制间质细胞增殖并增加平滑肌细胞表型表达(P<0.05、P<0.01);5μg/L的bFGF与0.001μg/L和0.01μg/L TGF-β1作用间质细胞,促进细胞增殖(P<0.01),与0.1μg/L,1μg/L及10μg/L TGF-β1作用间质细胞,抑制细胞增殖,0.1μg/L时对细胞的抑制作用轻微(P>0.05),1μg/L及10μg/L时出现明显的抑制(P<0.01),同时TGF-β1在较高浓度时(>1μg/L),平滑肌细胞表型表达明显增加(P<0.01)。结论:bFGF以时间和浓度依赖的方式促进培养的增生前列腺间质细胞的增殖,并减少平滑肌细胞表型表达;TGF-β1抑制间质细胞的生长并诱导间质细胞向平滑肌细胞分化,两者共同在BPH的形成机制中发挥着重要作用。     

联合应用bFGF和ATP对体外培养乳鼠脊髓神经元的影响

目的研究联合使用碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)和三磷酸腺苷(adenosinetri2phosphate,ATP)对体外培养的乳鼠脊髓神经元的作用。方法将不同的干预物加入培养基,分为bFGF组,ATP组,ATP bFGF组以及单纯对照组。相差倒置显微镜观察细胞生长情况,计数绘制细胞生长曲线,并测量细胞突起的长度;用MTT法测定培养细胞的存活率。结果(1)细胞突起长度:实验各组神经元轴突的长度均长于对照组(P<0.01)。接种48h和96h后,ATP bFGF组细胞突起长度明显长于ATP组(t=6.576、7.032,P<0.01);ATP bFGF组与bFGF组相比无差异(t=2.543、3.195,P>0.05);bFGF组细胞突起长度明显长于ATP组(t=6.245、8.026,P<0.01)。(2)比较MTT值:实验各组的灰度均明显大于对照组(P<0.01)。ATP bFGF组的灰度明显大于ATP组(t=6.053,P<0.01)。ATP bFGF组的灰度明显大于bFGF组(t=4.971,P<0.01)。bFGF组与ATP组相比无差异(P>0.05)。结论ATP、bFGF对于体外培养的脊髓神经元的存活均有较强的维持作用,并能促进轴突生长;两者联合使用作用明显增强。     

Cytokines produced by microwave-radiated Sertoli cells interfere with spermatogenesis in rat testis

Microwave radiation resulted in degeneration, apoptosis or necrosis in germ cells at different stages. The molecular mechanisms by which microwaves induce spermatogenesis disorder have not been completely understood. Sertoli cells play crucial roles in mammalian spermatogenesis. Cytokines produced by Sertoli cells play pleiotropic roles in different conditions. At physiologically low concentration, TNFα, IL-1β and IL-6 behave as survival factors; while under pathological condition, these cytokines can induce apoptosis in testis. The effects of cytokines produced by microwave-radiated Sertoli cells on spermatogenesis are poorly understood. The purpose of this study was to determine the effect of cytokines produced by microwave-radiated Sertoli cells on the germ cells. We focused the effect of TNFα, IL-1β and IL-6 on the germ cells. The results showed that TNFα, IL-1β and IL-6 were increased in Sertoli cells after exposure to microwave radiation. These up-regulated cytokines can induce apoptosis and lipid peroxidation in the membrane of germ cells. In addition, germ cell apoptosis was associated with the up-regulation of Bax/Bcl-2 and caspase-3. These results suggest that cytokines produced by microwave-radiated Sertoli cells may disrupt spermatogenesis. Our data provided novel insight into the injury mechanism of germ cells induced by microwave radiation.     

Epidermal growth factor stimulates lactate production and inhibits aromatization in cultured Sertoli cells from immature rats

The addition of epidermal growth factor (EGF) at nanomolar concentrations to cultures of Sertoli cells from 16-day-old rats induced more than a 2-fold stimulation of lactate production after 12 h of incubation. The effects of EGF, insulin and FSH on lactate production were very similar and the concentrations that produced half maximal stimulation were 11, 22 and 25 ng/ml or, in molar terms, 1.8, 3.5 and 0.6 nM for EGF, insulin and FSH, respectively. No synergistic or additive effects of these hormones on lactate production could be demonstrated. In contrast, EGF inhibited FSH-stimulated oestradiol synthesis by more than 50%. Insulin had no effect on FSH-stimulated aromatization.     

Evaluation of the ultrastructural changes in the human Sertoli cell in testicular disorders and the relationship of the changes to the levels of serum FSH

Sertoli cell ultrastructure was compared in men with testicular disorders (hypospermatogenesis; germ cell aplasia) and men with normal testes to determine if any specific cytological change could be correlated with diminished feedback from the testis resulting in elevated serum FSH levels. The normal Sertoli cell contained smooth endoplasmic reticulum, mitochondria and variable numbers of lipid inclusions, lipofuscin, crystals of Charcot-Böttcher and specialised inter-Sertoli cell junctional complexes. The principal abnormalities in Sertoli cells of men with testicular disorders were: 1) dilated vesicles of smooth endoplasmic reticulum and occasional expansions of the intercellular space; 2) increased numbers of cytoplasmic filaments; and 3) with germ cell aplasia inter-Sertoli cell junctions were complexly arranged due to interdigitation of Sertoli cell processes. Occasionally, increased lipid and lipofuscin aggregations were seen and in germ cell aplasia, aggregations of cytoplasmic glycogen were often present. Although these changes were seen more consistently with germ cell aplasia they were observed frequently with hypospermatogenesis where some tubules contained Sertoli cells with normal features. No correlation was found between abnormal Sertoli cell cytology and serum FSH levels.     

碱性成纤维细胞生长因子在大鼠BMSCs向睾丸Leydig细胞分化中的作用研究

目的 :研究碱性成纤维细胞生长因子(b FGF)在大鼠骨髓间充质干细胞(BMSCs)定向分化为睾丸Leydig细胞过程中的作用,探讨BMSCs定向分化为Leydig细胞的适宜方法。方法:将经纯化鉴定的SD大鼠BMSCs第3代细胞接种于6孔板,随机分为对照组(A)、人绒毛膜促性腺素(h CG)+血小板源性生长因子(PDGF)诱导组(B)、h CG+PDGF+5.0 ng/ml b FGF诱导组(C)、h CG+PDGF+10.0 ng/ml b FGF诱导组(D)、h CG+PDGF+20.0 ng/ml b FGF诱导组(E),分别于第7、14、21天进行形态学观察、培养液睾酮水平及3β-羟化类固醇脱氢酶(3β-HSD)免疫荧光检测。结果:B、C、D、E组细胞诱导后体积变大,互相连接呈长梭形或不规则形,贴壁生长,核大、较圆,符合Leydig细胞的特点。随着时间和b FGF浓度的增加,B、C、D、E组睾酮水平逐渐升高,3β-HSD阳性表达细胞逐渐增多,C、D、E组与B组,D、E组与C组间差异有统计学意义(P均<0.05);D、E组之间差异无统计学意义(P>0.05)。结论:b FGF在大鼠BMSCs向Leydig细胞体外诱导分化过程中有明显促进作用。     

温度对体外大鼠睾丸支持细胞增殖及occludin蛋白表达的影响

目的:通过观察不同温度对原代大鼠睾丸支持细胞增殖作用与紧密连接蛋白occludin(OCLN)表达的影响,探讨温度因素在男性生殖功能降低或男性不育中的作用机制。方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),油红O染色及免疫组化FasL鉴定。实验分为对照组(34℃)和实验组(35℃、36℃、37℃、38℃、39℃)。在相应的温度下培养4 d后,CCK-8法检测SC增殖作用,HE染色观察细胞形态及结构,Western印迹法和免疫荧光法检测OCLN表达水平。结果:体外培养SC的纯度为(96.20±1.95)%,CCK-8结果显示,细胞增殖活性在34～36℃时逐渐增强,36～39℃时逐渐降低,并在镜下观察发现细胞核固缩、裂解明显;免疫荧光及Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P<0.01)。结论:温度升高(>36℃)抑制SC的增殖活性,并降低SC紧密连接蛋白的表达量。因此,高温对SC活性和紧密连接完整性的影响,可能是导致男性生育能力下降或不育的机制之一。     

前列腺组织中EGF、bFGF的表达

目的：研究EGF、bFGF在前列腺组织中的表达。方法：应用mRNA斑点杂交、原位杂交、免疫组织化学及原位杂交与免疫组织化学双染法检测6例正常前列腺（NP）、27例良性前列腺增生症（BPH）前列腺组织中EGF及bFGF的表达。结果：BPH前列腺组织和NP组织中无均无EGF mRNA表达,EGF蛋白表达呈弱阳性,两组间差异无显著性意义（P＞0.05）;NP组织上皮细胞有较多bFGF mRNA表达,但无bFGF翻译,基底基质细胞有少量mRNA及蛋白表达,二者表达水平基本一致;BPH前列腺组织上皮细胞无bFGF mRNA表达,但局灶性增殖上皮细胞细胞膜上有bFGF,基底基质细胞有大量bFGF mRNA转录及蛋白质翻译,以局灶性增殖区最为明显。结论：NP及BPH的前列腺组织中无EGF分泌细胞;bFGF在前列腺基底基质细胞过度表达,以自分泌、旁分泌方式促进了基质和上皮的非均一性增殖。     

ADSC-bFGF对大鼠腹壁成形术后TRAM皮瓣活性的影响

目的：研究脂肪来源间充质干细胞复合碱性成纤维细胞生长因子（ADSC-bFGF）对大鼠腹壁成形术后腹部横行腹直肌肌皮瓣（TRAMflap）活性的影响。方法：体外分离、培养、鉴定SD大鼠ADSC。建立大鼠腹壁成形术模型。分别给予A组（ADSC-bFGF）、B组（ADSC）、C组（纤维蛋白凝胶）及D组（生理盐水）。所有动物腹壁成形术后一个月建立TRAM皮瓣模型。术后7天测定皮瓣成活面积、组织学观察和微血管密度（MVD）。结果：A、B、C、D四组的皮瓣存活率分别为（69.40±2.81）%、（66.19±2.32）%、（46.69±2.41）%和（43.77±3.55）%;A、B、C、D四组的毛细血管密度（根/mm2）分别为：（65.30±2.19）根/mm2;（59.99±2.41）根/mm2;（44.71±2.21）根/mm2;（42.44±3.22）根/mm2。皮瓣成活率A组与B组差异有意义（P〈0.05）,且这两组明显高于C组及D组,差异有显著性意义（P〈0.01）。微血管密度A组与B组差异有显著性意义（P〈0.01）,且这两组明显高于C组及D组,差异有显著性意义（P〈0.01）。免疫组化显示实验组中有大量褐色抗原抗体复合物沉积在血管内皮细胞胞浆内。结论：ADSC-bFGF能刺激大鼠腹部横行腹直肌肌皮瓣的血管新生和增加血管数量,从而增加皮瓣的存活率。     

Method for evaluating quality of cultured neonatal pig Sertoli cells

BACKGROUND: Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two-step enzymatic digestion with limited assessment of the culture. Here we describe a one-step method, and a series of tests for determining purity, viability, and function of the cultured cells. METHODS: We isolated SC from neonatal pigs using Liberase HI digestion. Viability and apoptosis of cultured cells were measured by flow cytometry with propidium iodide and annexin, respectively. Specific identification of the Sertoli type was made by immunodetection of Sox9, vimentin, and Mullerian inhibiting substance. Moreover, for functionality we were able to detect clusterin in the cultured cells by Western blot. RESULTS: Our isolation method had a yield and purity similar to previous reports measured with two-step methods. Viability was 95.22 +/- 0.57% and apoptotic cells were 10.5 +/- 0.32% after 48 h in culture. At 7 days, practically all cells expressed Sox9, Mullerian inhibiting substance, clusterin, and vimentin. CONCLUSIONS: We describe an alternative strategy for preparing and identifying cultured SC for further assays of metabolic activity or in transplantation models. Establishing a one-step Liberase-digestion method for isolation, evaluating viability and apoptosis by more sensitive methods, and detecting specific markers in culture can help to evaluate the quality of cultured cells. Specific cell markers for identifying SC may be critical when identifying SC outside the testis, in contrast with vimentin which is useful only for in situ cells.