共培养诱导骨髓间充质干细胞向leydig细胞分化的研究
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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。
Background
Testes loss caused by trauma or surgical procedure for tumor treatment was not rare, and the morbidity of hypoandrogenism has increasing recently as a result of congenital disease, environmental pollution, anabatic working pressure and an increased aging population. However, until now there was no reliable and effective therapic method for this kind of diseases. With the development of tissue engineering technology, construction of tissueengineered androgen-secreting tissue seems promising therapic method in treating hypoandrogenism or testes loss. Using Leydig cell as seed cells, the tissue-engineered androgen-secreting tissue could secrete testosterone under the control of the pituitary-gonad axis, thus avoided the adverse effect commonly followed soly hormone therapy. Another advantage of androgen-secreting tissue was the plasticity of its shape, which could be molded like a testes shape that could fulfill the patient’s strongly psychological and social need.
The limited source of seed cells had greatly hampered the further study and clinical perspective of tissue-engineered androgen-secreting tissue study. Bone Mesenchymal Marrow Cells (BMCs) contain several pluripotent progenitor cells, which differentiate into multiple lineages. It can be isolated from bone marrow aspirated by preferential adherence to tissue culture plastic or by differential centrifugation through a percoll or ficoll density gradient to obtain a nucleated cell fraction. Pittenger et al. showed that MSCs represent only 0.001–0.01% of the total number of nucleated cells in the bone marrow; however, this population can be expanded ex vivo and enriched by standard culture conditions. So BMSCs may provide new source for seed cells in androgen secretiong tissue construction. 2006 Takashi Yazawa, etl shows that by adenovirus -mediated forced expression of SF-1, it could transform mouse primary long-term cultured BMCs into steroidogenic cells. 2007 year, YanHe Lue, Transplanted Bone Marrow Stem Cells into the Testis, and find adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages and author indicate that the resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. So,it provide theoretical basis for co-culture of leydig cell and BMSCs in vitro。
These study results above-mentioned shows that the feasibility of the availability of leydig cell indued by BMSCs, however, it maybe a long way to go when come to clininc applicaton. The technical of transfected BMSCs by adenovirus may obtain a new source of a secretory cell of androgen hormone, but the potential problem of biological safety followed adenovirus transferction is worldwide obstacle, and may not be solved in near future. Transplation of BMSCs into testis is way of experimental study; the high qualification of recipient was needed. For low proliferation of leydig cell in patients with hypoandrogenism, the opportunity of the practical application is slim. The initial factors and mechanism for the differentiation of stem or progenitor cells to leydig are not clear; however, by the way of imitation of microenvironment of leydig cell, it is feasible to induce BMSCs to differentiate into leydig cell theropially.Not only a promising way to gain the sufficient seed cells for tissue reconstruction for thus kinds of dieases, but also a new way have been provided to investigate the detail mechanisms under the differentiation of stem or progenitor leydig cell to mature leydig cell in the process of testis development by this study.
Objective
To provide a new source for seed cell for tissue-engineered androgen secretiong tissue, we investigate the possibility of indued the BMSCsto differentiate into leydig cell by co-culture system with transwell culture equipment. We contrived a simple and quick technique for LC isolation via differential adhesion. This method had increased LC number recovered from the testes, reduced cell damage during isolation. Bone marrow cells obtained from a nucleated cell fraction which by gradient solution centrifugation through a Ficoll density gradient. The mononuclear cell fraction obtained at the interface is explanted ex vivo by plating. And in 10%FBS DMEM medium, it proliferates quickly, and passage 3 cells were obtained to co-culture with leydig cells. After 2 and 4 weeks co-culture, BMSCs were accept immunohistochemistry and RT-PCR analysis, several specific marker of leydig cell were contained.
Methods
1.Identification,isolation and culture of Bone mesenchymal cells: BMSCs were harvested from femurs of adult wistar rats aged 3weeks by flushing the shaft with Dulbecco’s Modified Eagle’s Medium (DMEM) using a syringe with a 26-gauge needle. The aspirate was washed with phosphate buffered saline (PBS) with 2% fetal bovine serum, and then separated by Ficoll density gradient centrifugation to obtain mononuclear cells. After 4 day, nonadherent cells were removed, and adherent cells were thoroughly washed twice with PBS. Medium was replaced every 3–4 days. After 6 days culture, the colony-forming unit fibroblastic,CFU-Fs were formed. 2 weeks after cultured by the sepecial indued culture medium, BMSCs differentiate into adipocytes, and osteogenic cells.
2.Isolation of leydig cell by differential adhesion: 3w old rats obtained by differential adhesion as discribed above, 3β-HSD and LHR expression in cultured cells were tested by immunohistochemistry 1 day culture. Testosterone productivity in isolated Leydig cells with or without HCG stimulation were also tested. Testes of 3w old Wister rats were harvested, after collagenase digestion, the seperated cells were filtrated by 33μm stainlesss-teel mesh, centrifuged and then cultured for 2 hours. After 2 hours’selective adhesion, the superanant was discarded, the adherent cells were than washed with PBS, and cultured under standard condition.
3. In vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: leydig cell and BMSCs from 3w old rats were obtained by differential adhesion and Ficoll density gradient centrifugation, the cells were co-cultured in DMEM/F12 media with 3% FBS, Medium was replaced every 1–2 days .2 weeks after co-culture, 3β-HSD and LHR expression of BMSCS were tested by immunohistochemistry and the mRNA transcription of several key steroid genetic synthetases were examined by RT-PCR. The results show that BMSCs express the LHR, and the mRNA of stAR、3β-HSD and LHR, were transcripted after 2 weeks co-cultivation.
Results
PartⅠIdentification,isolation and culture of Bone mesenchymal cells: BMSCs from 3w old rats were obtained by Ficoll density gradient centrifugation and adhesion. After 6 days culture, the colony-forming unit fibroblastic (CFU-Fs) were formed, 2 weeks after cultured by the special inducing culture medium, BMSCs differentiate into adipocytes, and osteogenic cells. The ability of the forming of CFU-Fs and differentiation of adipocytes, and osteogenic cells, shows that the cells gained from this method can self-renew and be capacable of differentiate into at least one high differentiated cell.
PartⅡIsolation of leydig cell by differential adhesion: Leydig cell of 3w old rats can be obtained by differential adhesion together with administration contain: collagenase digestion, filtration with 33μm mesh, centrifugalization and After 2 hours’selective adhesion. After culture 1 day, the cells were test by 3β-HSD and LHR immunohistochemistry. It shows that most of all cells cultured were positive for 3β-HSD and LHR. So it is confirm that it is effective way to gain leydig cells with high purity, and can be used as cell in co-cultivation system.
PartⅢIn vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: Both Leydig cells and proliferated BMSCs showed good compatibility with in transwell co-cultured system with medium of DMEM/F12 with 3%FBS.2 weeks later, BMSCs were show positive for LHR and 3β-HSD, and is mRNA also tested by RT-PCR。So the co-culture system is a new way to induce BMSCs to differentiate into leydig-like cell.
Conclusion
This study primitively proved that BMSCs can be induced to differentiate into leydig-like cells, for 3β-HSD and LHR both two specific marker for leydig cell.were ditected in BMSCs after co-cultured for 2 and 4 weeks; secondly, we testify Leydig cell lineage, could be successfully obtained by differential adhesion. In the further studys, We will add several growth factors or neutrition materials which were needed during leydig cell differentiation, and investigate the mechanism of differentiation of leydig stem cell to adult leydig cell。We will find safe and effective way to obtain the proper seed cells for constructed tissue-engineered androgen secretiong tissue or hormone substitution in deficiency of androgen secretiong tissue study, and may provide a promising method for the diseases of shortage of testerone.
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。
Background
Testes loss caused by trauma or surgical procedure for tumor treatment was not rare, and the morbidity of hypoandrogenism has increasing recently as a result of congenital disease, environmental pollution, anabatic working pressure and an increased aging population. However, until now there was no reliable and effective therapic method for this kind of diseases. With the development of tissue engineering technology, construction of tissueengineered androgen-secreting tissue seems promising therapic method in treating hypoandrogenism or testes loss. Using Leydig cell as seed cells, the tissue-engineered androgen-secreting tissue could secrete testosterone under the control of the pituitary-gonad axis, thus avoided the adverse effect commonly followed soly hormone therapy. Another advantage of androgen-secreting tissue was the plasticity of its shape, which could be molded like a testes shape that could fulfill the patient’s strongly psychological and social need.
The limited source of seed cells had greatly hampered the further study and clinical perspective of tissue-engineered androgen-secreting tissue study. Bone Mesenchymal Marrow Cells (BMCs) contain several pluripotent progenitor cells, which differentiate into multiple lineages. It can be isolated from bone marrow aspirated by preferential adherence to tissue culture plastic or by differential centrifugation through a percoll or ficoll density gradient to obtain a nucleated cell fraction. Pittenger et al. showed that MSCs represent only 0.001–0.01% of the total number of nucleated cells in the bone marrow; however, this population can be expanded ex vivo and enriched by standard culture conditions. So BMSCs may provide new source for seed cells in androgen secretiong tissue construction. 2006 Takashi Yazawa, etl shows that by adenovirus -mediated forced expression of SF-1, it could transform mouse primary long-term cultured BMCs into steroidogenic cells. 2007 year, YanHe Lue, Transplanted Bone Marrow Stem Cells into the Testis, and find adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages and author indicate that the resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. So,it provide theoretical basis for co-culture of leydig cell and BMSCs in vitro。
These study results above-mentioned shows that the feasibility of the availability of leydig cell indued by BMSCs, however, it maybe a long way to go when come to clininc applicaton. The technical of transfected BMSCs by adenovirus may obtain a new source of a secretory cell of androgen hormone, but the potential problem of biological safety followed adenovirus transferction is worldwide obstacle, and may not be solved in near future. Transplation of BMSCs into testis is way of experimental study; the high qualification of recipient was needed. For low proliferation of leydig cell in patients with hypoandrogenism, the opportunity of the practical application is slim. The initial factors and mechanism for the differentiation of stem or progenitor cells to leydig are not clear; however, by the way of imitation of microenvironment of leydig cell, it is feasible to induce BMSCs to differentiate into leydig cell theropially.Not only a promising way to gain the sufficient seed cells for tissue reconstruction for thus kinds of dieases, but also a new way have been provided to investigate the detail mechanisms under the differentiation of stem or progenitor leydig cell to mature leydig cell in the process of testis development by this study.
Objective
To provide a new source for seed cell for tissue-engineered androgen secretiong tissue, we investigate the possibility of indued the BMSCsto differentiate into leydig cell by co-culture system with transwell culture equipment. We contrived a simple and quick technique for LC isolation via differential adhesion. This method had increased LC number recovered from the testes, reduced cell damage during isolation. Bone marrow cells obtained from a nucleated cell fraction which by gradient solution centrifugation through a Ficoll density gradient. The mononuclear cell fraction obtained at the interface is explanted ex vivo by plating. And in 10%FBS DMEM medium, it proliferates quickly, and passage 3 cells were obtained to co-culture with leydig cells. After 2 and 4 weeks co-culture, BMSCs were accept immunohistochemistry and RT-PCR analysis, several specific marker of leydig cell were contained.
Methods
1.Identification,isolation and culture of Bone mesenchymal cells: BMSCs were harvested from femurs of adult wistar rats aged 3weeks by flushing the shaft with Dulbecco’s Modified Eagle’s Medium (DMEM) using a syringe with a 26-gauge needle. The aspirate was washed with phosphate buffered saline (PBS) with 2% fetal bovine serum, and then separated by Ficoll density gradient centrifugation to obtain mononuclear cells. After 4 day, nonadherent cells were removed, and adherent cells were thoroughly washed twice with PBS. Medium was replaced every 3–4 days. After 6 days culture, the colony-forming unit fibroblastic,CFU-Fs were formed. 2 weeks after cultured by the sepecial indued culture medium, BMSCs differentiate into adipocytes, and osteogenic cells.
2.Isolation of leydig cell by differential adhesion: 3w old rats obtained by differential adhesion as discribed above, 3β-HSD and LHR expression in cultured cells were tested by immunohistochemistry 1 day culture. Testosterone productivity in isolated Leydig cells with or without HCG stimulation were also tested. Testes of 3w old Wister rats were harvested, after collagenase digestion, the seperated cells were filtrated by 33μm stainlesss-teel mesh, centrifuged and then cultured for 2 hours. After 2 hours’selective adhesion, the superanant was discarded, the adherent cells were than washed with PBS, and cultured under standard condition.
3. In vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: leydig cell and BMSCs from 3w old rats were obtained by differential adhesion and Ficoll density gradient centrifugation, the cells were co-cultured in DMEM/F12 media with 3% FBS, Medium was replaced every 1–2 days .2 weeks after co-culture, 3β-HSD and LHR expression of BMSCS were tested by immunohistochemistry and the mRNA transcription of several key steroid genetic synthetases were examined by RT-PCR. The results show that BMSCs express the LHR, and the mRNA of stAR、3β-HSD and LHR, were transcripted after 2 weeks co-cultivation.
Results
PartⅠIdentification,isolation and culture of Bone mesenchymal cells: BMSCs from 3w old rats were obtained by Ficoll density gradient centrifugation and adhesion. After 6 days culture, the colony-forming unit fibroblastic (CFU-Fs) were formed, 2 weeks after cultured by the special inducing culture medium, BMSCs differentiate into adipocytes, and osteogenic cells. The ability of the forming of CFU-Fs and differentiation of adipocytes, and osteogenic cells, shows that the cells gained from this method can self-renew and be capacable of differentiate into at least one high differentiated cell.
PartⅡIsolation of leydig cell by differential adhesion: Leydig cell of 3w old rats can be obtained by differential adhesion together with administration contain: collagenase digestion, filtration with 33μm mesh, centrifugalization and After 2 hours’selective adhesion. After culture 1 day, the cells were test by 3β-HSD and LHR immunohistochemistry. It shows that most of all cells cultured were positive for 3β-HSD and LHR. So it is confirm that it is effective way to gain leydig cells with high purity, and can be used as cell in co-cultivation system.
PartⅢIn vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: Both Leydig cells and proliferated BMSCs showed good compatibility with in transwell co-cultured system with medium of DMEM/F12 with 3%FBS.2 weeks later, BMSCs were show positive for LHR and 3β-HSD, and is mRNA also tested by RT-PCR。So the co-culture system is a new way to induce BMSCs to differentiate into leydig-like cell.
Conclusion
This study primitively proved that BMSCs can be induced to differentiate into leydig-like cells, for 3β-HSD and LHR both two specific marker for leydig cell.were ditected in BMSCs after co-cultured for 2 and 4 weeks; secondly, we testify Leydig cell lineage, could be successfully obtained by differential adhesion. In the further studys, We will add several growth factors or neutrition materials which were needed during leydig cell differentiation, and investigate the mechanism of differentiation of leydig stem cell to adult leydig cell。We will find safe and effective way to obtain the proper seed cells for constructed tissue-engineered androgen secretiong tissue or hormone substitution in deficiency of androgen secretiong tissue study, and may provide a promising method for the diseases of shortage of testerone.
引文
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2. Theodoraki A, Bouloux PM. Testosterone therapy in men. Menopause Int. 2009; 15(2):87-92.
3. Midzak AS, Chen H, Papadopoulos, et al. Leydig cell aging and the mechanisms of reduced testosterone synthesis. Mol Cell Endocrinol. 2009;299(1):23-31
4. Atala A. Tissue engineering approaches for genital reconstruction. Adv Exp Med Biol. 2002; 511:289-303
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6. Gondo S, Okabe T, Tanaka T, et al. Adipose tissue-derived and bone marrow-derived mesenchymal cells develop into different lineage of steroidogenic cells by forced expression of steroidogenic factor 1. Endocrinology. 2008;149(9):4717-25
7.张思孝,董强.睾丸Leydig细胞干细胞研究进展.中华男科学杂志. 2005; 11 (11): 803-805
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9. Yazawa T, Inanoka Y, Mizutani T, Kuribayashi M, Umezawa A, Miyamoto K. Liver Receptor Homolog-1 Regulates the Transcription of Steroidogenic Enzymes and Induces the Differentiation of Mesenchymal Stem Cells into Steroidogenic Cells. Endocrinology 2009; 150 (8): 3885-93.
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11. Lue Y, Erkkila K, Liu PY, et al. Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure. Am J Pathol. 2007;170(3):899-908.
12. Siril Ariyaratne HB, Chamindrani Mendis-Handagama SML, Hales DB, et al. Studies on the Onset of Leydig Precursor cell differentiation in the prepubertal rat testis.Biology of reproduction. 2000; 63: 165-171
13. Gondo S, Yanase T, Okabe T, et al. SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells. Genes Cells 2004(9):1239-1247
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