共培养诱导骨髓间充质干细胞向leydig细胞分化的研究

发布时间：2018-04-16 06:25

本文选题：骨髓间充质干细胞 + Leydig细胞　；参考：《第二军医大学》2011年博士论文

【摘要】：研究背景随着环境污染、人口老龄化的日益加重,雄激素缺乏性疾病的发病率具有逐年上升的趋势,雄激素的缺乏不但严重影响患者身体健康如男性第二性征异常、造成生理功能紊乱,且严重影响男性的心理健康,容易造成社会问题[1、2]。针对这类疾病,传统上的治疗主要靠外源性的雄性激素补充或替代,但由于外源性雄激素补充无法接受垂体-性腺轴的生理调节,易造成体内激素失调失调,且抑制体内的正常雄性激素的分泌,长期使用常会引起高血压、红细胞增多、骨密度异常等一系列严重并发症[2]。与之相比,Leydig细胞移植,可接受下丘脑-垂体-性腺轴的调节,符合人的生理规律,具有明显优势,是治疗雄激素缺乏性疾病的可靠、理想的治疗途径[5]。随着组织工程技术的发展,采用组织工程技术以Leydig细胞作为种子细胞,重建具有正常生理功能和形态的组织,则有望在形态、功能两个方面解决睾丸缺失/雄激素缺乏性疾病,为该类疾病的治疗提供更符合生理的治疗途径[4]。虽然Leydig细胞治疗具有明显的优势,但细胞来源不足和免疫排斥问题是制约该技术发展的主要瓶颈。Leydig细胞仅存于睾丸,数量有限(仅占睾丸细胞总量的2-5%),普遍依靠同种异体供应,存在免疫排斥问题,临床广泛应用受到极大限制[5]。来源于自体骨髓组织或脂肪组织的间充质干细胞,取材方便,通过体外分离、培养及富集,可获得大量的细胞,如果能将其诱导成leydig细胞,则可以同时解决leydig细胞移植的来源及免疫排斥两个问题。普遍认为,性腺及肾上腺等类固醇合成器官从来源于中段中胚层的肾上腺生殖器原基发育而来[6],而Leydig细胞来源于胚胎发育期的中肾胚的间质细胞,并认为位于管周的成纤维样细胞为leydig干细胞或前体细胞,leydig细胞由这些细胞分化而来[7]。间充质干细胞(mesenchymal Stem Cells,MSCs)与leydig干细胞都来源于中胚层,因而具有向Leydig细胞转化的理论可能[8]。普遍认为类固醇因子-1(steroidogenic factor 1, SF-1)是睾丸及肾上腺发育及类固醇激素合成的关键的核内受体,在它的调控下,类固醇合成酶的各个基因得以表达[6]。Tomoko Tanaka在2006年用骨髓基质细胞注入发育期的大鼠睾丸内,这些细胞表现出与leydig细胞相似的特性;同时将携带绿色荧光蛋白的P450scc用腺病毒携带AD/4Bp基因后转染体外分离小鼠BMSCs,并在加入cAMP培养液中培养2周后,这些细胞表现出leydig细胞的特性,并能分泌睾酮[8]。Gondo等在2008年用这种方法处理BMSCs及脂肪干细胞(adipose-derived mesenchymal stem cells, AMSCs)后,发现BMSCs向产生睾酮的leydig细胞方向分化,而AMSCs则倾向于产生糖皮质类激素的肾上腺细胞分化[6]。Takashi2009年将肝受体同系物-1(liver receptor homolog-1,LRH)以质粒转染人的BMSCs及cAMP处理后,这些细胞表达肾上腺及睾酮合成的基因[9]。该类实验表明BMSCs在异位表达的SF-1的刺激下可以向leydig细胞方向转化,转录并表达睾酮合成过程中所需的各种酶,并具有睾酮分泌功能。该类实验主要用于研究性腺器官的发育,由于腺病毒转染的潜在安全问题,诱导后的细胞在临床治疗中的意义不大,同时说明在诱导分化为leydig细胞上BMSCs比ADSCs更适合。另外的研究主要是利用leydig细胞在体内生长的微环境在也可将BMSCs诱导向leydig细胞方向分化。Takashi Yazawa及Tomoko Tanaka等将大鼠BMSCs注射入3周龄发育期大鼠睾丸,结果显示注射的部分BMSCs能向Leydig细胞分化,并表达leydig细胞的各种细胞标志物[8、10]。YanHe Lue等进一步探讨了BMSCs注射入睾丸的曲精小管及睾丸间质的分化结局,绿色荧光蛋白标记的BMSCs分别注射入正常leydig细胞的大鼠及c-kit基因沉默的W/Wv大鼠(睾丸发育缺陷,无法形成精原细胞)睾丸内,10-12周后注射入正常大鼠的睾丸内BMSCs根据邻近细胞的情况,依次分化为与邻近细胞相识的Leydig细胞、Sertoli细胞、生殖细胞系细胞等体细胞成分,而注射入睾丸发育畸形鼠体内的BMSCs未发生分化。虽该然不能完全排除细胞融合的可能,但是可以说明睾丸各种细胞生长的微环境对移植BMSCs的定向分化具有诱导作用[11]。上述研究成果显示BMSCs诱导分化成为Leydig细胞具有可行性,但距临床应用仍有差距。基因转染技术虽然有望在体外大量的诱导BMSCs分化为雄激素分泌细胞,但该方法必须克服腺病毒转染技术带来的生物安全性问题,目前尚属世界性难题。BMSCs睾丸内注射属实验性研究,要求受体动物在发育期,对于Leydig细胞功能不足或不存在睾丸的受体意义不大,且分化的效率不高,目前通过该途径很难获得大量的自体BMSCs分化的Leydig细胞。然而,以上研究成果提示,虽然目前对睾丸分化微环境促进BMSCs定向分化的始动因素并不明确,但通过体外模拟Leydig细胞生长的微环境,在体外实现BMSCs向Leydig细胞大量的定向分化具备理论可能性。综上所述,针对Leydig细胞治疗及雄激素分泌组织构建研究中关键的种子细胞来源问题,结合本课题组前期实验中,通过差速贴壁法可以大量获得较纯的leydig细胞的基础上,将骨髓间充质干细胞与Leydig细胞共培养,利用leydig细胞生长的微环境及分泌的可溶性因子诱导BMSCs向Leydig细胞分化,以解决种子细胞来源不足的难题。该项研究可为临床雄性激素缺乏症提供一条安全有效的途径,同时对于进一步研究胚胎发育过程中leydig干细胞具体分化过程中的各种细胞因子的作用及调控机制具有借鉴意义。研究目的本研究从骨髓基质干细胞开始,通过Ficoll液分离、贴壁、增殖并传代,富集足够的BMSCs,并经过诱导分化成骨、成脂肪鉴定BMSC的多向分化能力;通过差速贴壁法获得的大鼠睾丸leydig细胞的免疫组化分析,证实差速贴壁法可以获得较纯的大鼠睾丸leydig细胞;探索共培养体系对BMSC向leydig细胞诱导的可能性,并对诱导后的BMSC进行leydig细胞的各项特异性指标进行免疫组化、RT-PCR的测定,检验诱导的BMSC作为雄激素分泌组织种子细胞的可行性,为解决组织工程化雄激素分泌组织研究中种子细胞来源不足的问题提供有效手段。研究方法一、密度梯度离心法获得骨髓基质干细胞及鉴定: 3周龄大鼠的股骨、胫骨,10ml注射器冲洗,1.083g/ml的ficoll去除血细胞,2天后半量换液,4天后首次全部换液,获取的贴壁细胞,并传代培养。取第三代BMSC进行成骨、成脂肪分化诱导。同时观察细胞形态,克隆样集落形成,并检测细胞增殖情况。二、差速贴壁法分离纯化3周鼠龄大鼠Leydig细胞:取3周龄雄性Wister大鼠双侧睾丸,无菌下去除白膜及较大的血管,用胶原酶震荡消化和400目(Ф33μm)不锈钢滤网过滤,离心后贴壁培养2小时,弃上清并漂洗3次,加入3%FBS的DMEM/F12培养液培养。三、共培养体系对BMSC诱导分化作用:3周龄大鼠睾丸leydig分离后以transwell培养皿双层间接共培养体系进行培养,隔1-2天换液,2周、4周后对诱导的细胞3β-HSD、LHR免疫化学染色、免疫荧光染色,PCR检测stAR、3β-HSD及LHR的表达情况及睾酮分泌水平的测定。研究结果第一部分梯度密度离心法获得骨髓基质干细胞及鉴定通过剥离股骨、胫骨,10ml注射器冲洗洗骨髓,1.083g/ml的Ficoll液去除红细胞,离心后用10%小牛血清的低糖DMEM培养液接种,贴壁4天后首次全量换液,可以获取的数量不等的贴壁细胞,并形成克隆集落样生长,传代培养后细胞形态、生长能力无明显变化,3天后即可观察到可见BMSC呈的克隆集落样生长,在体外培养条件下可以传至6-7代。取第三代的BMSC可以诱导向成骨、成脂肪分化,说明获得的BMSCs具备很强的增殖能力,并多向诱导分化,可用于下一步研究。第二部分差速贴壁法获得的LC细胞成分分析采用差速贴壁法能够获得3周龄大鼠睾丸LC细胞,经3β-HSD、LHR免疫化学染色分析,结果显示在DMEM/F12培养体系内培养1天后,几乎所有的细胞都表达3β-HSD、LHR抗体,提示我们该方法能有效对leydig细胞进行分离、富集。第三部分leydig细胞与BMSCs的共培养体系对BMSC诱导分化作用的研究通过3周鼠龄的leydig细胞与BMSCs间接共培养2周、4周后,免疫组化、PCR分析鉴定共培养的BMSCs情况,免疫组化显示部分BMSC表达leydig细胞特异性标志物3β-HSD、LHR, RT-PCR反应检测到leydig细胞的部分特异性标志物stAR、3β-HSD及LHR的mRNA表达,说明在间接共培养条件下BMSC可以向leydig样细胞分化。研究结论本研究证实差速贴壁法能够有效获得大鼠睾丸Leydig系细胞的各级细胞成分,通过间接共培养体系,能够诱导骨髓间质干细胞向leydig样细胞方向上分化,并表达leydig细胞的特异性标志物。我们推测通过进一步分析leydig细胞分化的始动因素及leydig细胞分化过程中的各种所需的各种营养因子,优化实验培养条件将可能诱导BMSCs分化为成熟的leydig细胞,为临床上雄性激素缺乏性疾病提供一条理想的治疗途径。
[Abstract]:Background of the study

With the increasing of environmental pollution and aging population , the incidence of androgen deficiency disease has been increasing year by year .

In view of this kind of disease , the traditional therapy mainly depends on exogenous male hormone replacement or substitution , but because exogenous androgen supplement cannot accept the physiological regulation of the pituitary - sex gland axis , it is easy to cause the imbalance of hormone in the body and inhibit the secretion of normal male hormone in the body , and the long - term use of regular session causes a series of serious complications such as hypertension , red blood cell increase and bone mineral density . Compared with it , it can be accepted the regulation of hypothalamus - pituitary - sex gland axis , which accords with the physiological law of human , and has obvious advantages . It is a reliable and ideal treatment route for the treatment of androgen deficiency disease . With the development of tissue engineering technology , tissue engineering technology is used as seed cell to reconstruct tissues with normal physiological function and morphology .

Although it has obvious advantages , the problem of cell - derived insufficiency and immune rejection is the main bottleneck which restricts the development of the technology . The cells only exist in the testis , the number is limited ( only 2 - 5 % of the total amount of testis cells ) .

The origin and immune rejection of leydig cells derived from autologous bone marrow tissue or adipose tissue can be solved at the same time . Mesenchymal stem cells ( MSCs ) and leydig stem cells are all derived from mesoderm , and therefore the theory of transforming to dig cells is likely to be about 8 million . It is widely recognized that steroidogenic factor 1 ( SF - 1 ) is the key nuclear receptor for the development of testis and adrenal gland and steroid hormone synthesis . Under its control , each gene of steroid synthetase is expressed as a steroid hormone . In the testis of rats injected with bone marrow stromal cells into the developmental stage in 2006 , the cells showed similar characteristics to leydig cells , and then transfected with the AD / 4Bp gene by adenovirus carrying the green fluorescent protein , and then transfected into the in vitro isolated mice , and cultured for 2 weeks in the addition of cAMP medium , the cells showed the characteristics of leydig cells , and the testosterone propionate was secreted . Gondo et al . , after treated with this method in 2008 , and adipose - derived mesenchymal stem cells ( AMSCs ) , found that they differentiated into leydig cells producing testosterone , while AMSCs tended to differentiate between adrenal cells producing glucocorticoid - like hormones . In 2009 , the liver receptor homologue - 1 ( LRH ) was transfected into human bone marrow cells and cAMP treated by plasmid - transfected cells , and the genes expressed by these cells expressed adrenal gland and testosterone synthesis . The experiments indicated that the cells could be transformed into leydig cells under the stimulation of ectopic expression of SF - 1 , transcription and expression of various enzymes needed in the synthesis of testosterone , and had the function of testosterone secretion .

The experimental results showed that bone marrow cells were injected into the testes of rats with normal leydig cells , and the cells were injected into normal leydig cells . The results showed that bone marrow cells were injected into normal leydig cells , and the cells were injected into the testis of normal rats .

However , it is difficult to obtain a large amount of tumor cells in vitro . However , it is difficult to obtain a large number of cells in vitro . However , it is difficult to obtain a large amount of autologous bone marrow stromal cells in vitro .

In conclusion , according to the problem of seed cell origin in the research of the treatment and androgen secretion , the bone marrow mesenchymal stem cells can be cultured in a large amount by means of differential wall method , and the bone marrow mesenchymal stem cells can be co - cultured with each other . The research can provide a safe and effective way for clinical male hormone deficiency , and can be used for further research on the role of various cytokines in the specific differentiation of leydig cells in the process of embryo development and the mechanism of regulation and regulation .

Purpose of study

In this study , the bone marrow stromal cells were isolated , adherent , proliferated and passaged through Ficoll solution , which was enriched in bone and fat to identify the multi - directional differentiation ability of BMSC . By means of differential adherent method , the rat testis leydig cells could be obtained . The feasibility of the co - culture system on leydig cells was investigated . The feasibility of the induction of BMSC as androgen secretion tissue seed cells was investigated .

Research Methods

1 . Bone marrow stromal cells were obtained by density gradient centrifugation . The bone marrow stromal cells were washed with 1 . 08 g / ml ficoll , 1 . 08 g / ml ficoll removed blood cells , 1 . 08 g / ml ficoll removed the blood cells , 2 days later , the cells were completely changed and the adherent cells were cultured .

3 weeks old male Wister rats were isolated and purified by differential wall method . The male Wister rats were divided into three weeks old male Wister rats , then the white film and the larger blood vessels were removed . After centrifugation , the cells were cultured in DMEM / F12 medium supplemented with 3 % FBS for 2 hours , discarded supernatant and rinsed 3 times .

3 . The effect of co - culture system on BMSC - induced differentiation : 3 - week - old rat testis leydig was isolated and cultured in a double - layer indirect coculture system of transwell culture dish . After 1 - 2 days of change , 3 尾 - HSD , LHR immunochemical staining , immunofluorescence staining and PCR were used to detect the expression of stAR , 3尾 - HSD and LHR and the level of testosterone secretion .

Results of the study

Bone marrow stromal cells were obtained from bone marrow stromal cells by the first partial gradient density centrifugation . Red blood cells were washed with 1 . 08 g / ml Ficoll solution by washing bone marrow and 1.083 g / ml Ficoll solution . After centrifugation , the cells were inoculated with 10 % calf serum for the first time . After 3 days , the colonies could be transferred to 6 - 7 generations .

Analysis of the Components of LC Cells Obtained by the Second Partial Differential Wall - Wall Method

Three - week - old rat testis LC cells were obtained by differential adherent method . The results showed that after cultured in DMEM / F12 culture system for 1 day , almost all the cells expressed 3尾 - HSD , LHR antibody , suggesting that the method can effectively separate and enrich leydig cells .

The third part leydig cells and the co - culture system of BMSC induced the differentiation of BMSC . After 4 weeks , immunohistochemistry and PCR analysis were used to identify the co - cultured BMSC . The specific markers stAR , 3尾 - HSD and LHR mRNA expression of leydig cells were detected by immunohistochemistry and RT - PCR .

Conclusions of the study

This study demonstrated that the differential adherent method can effectively obtain the cellular components at all levels of rat ' s rat ' s rat testis , and can induce the differentiation of bone marrow mesenchymal stem cells in the direction of leydig - like cells and express leydig cells ' specific markers by indirect coculture . We surmise that by further analyzing the initial factors of leydig cell differentiation and the various nutrient factors needed in leydig cell differentiation , it is possible to optimize the experimental culture conditions to differentiate into mature leydig cells and provide an ideal treatment route for clinical male hormone deficiency diseases .

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

【参考文献】