發(fā)布時(shí)間：2018-04-16 06:25

  本文選題：骨髓間充質(zhì)干細(xì)胞 + Leydig細(xì)胞�。� 參考：《第二軍醫(yī)大學(xué)》2011年博士論文

【摘要】：研究背景 隨著環(huán)境污染、人口老齡化的日益加重,雄激素缺乏性疾病的發(fā)病率具有逐年上升的趨勢(shì),雄激素的缺乏不但嚴(yán)重影響患者身體健康如男性第二性征異常、造成生理功能紊亂,且嚴(yán)重影響男性的心理健康,容易造成社會(huì)問題[1、2]。 針對(duì)這類疾病,傳統(tǒng)上的治療主要靠外源性的雄性激素補(bǔ)充或替代,但由于外源性雄激素補(bǔ)充無法接受垂體-性腺軸的生理調(diào)節(jié),易造成體內(nèi)激素失調(diào)失調(diào),且抑制體內(nèi)的正常雄性激素的分泌,長期使用常會(huì)引起高血壓、紅細(xì)胞增多、骨密度異常等一系列嚴(yán)重并發(fā)癥[2]。與之相比,Leydig細(xì)胞移植,可接受下丘腦-垂體-性腺軸的調(diào)節(jié),符合人的生理規(guī)律,具有明顯優(yōu)勢(shì),是治療雄激素缺乏性疾病的可靠、理想的治療途徑[5]。隨著組織工程技術(shù)的發(fā)展,采用組織工程技術(shù)以Leydig細(xì)胞作為種子細(xì)胞,重建具有正常生理功能和形態(tài)的組織,則有望在形態(tài)、功能兩個(gè)方面解決睪丸缺失/雄激素缺乏性疾病,為該類疾病的治療提供更符合生理的治療途徑[4]。 雖然Leydig細(xì)胞治療具有明顯的優(yōu)勢(shì),但細(xì)胞來源不足和免疫排斥問題是制約該技術(shù)發(fā)展的主要瓶頸。Leydig細(xì)胞僅存于睪丸,數(shù)量有限(僅占睪丸細(xì)胞總量的2-5%),普遍依靠同種異體供應(yīng),存在免疫排斥問題,臨床廣泛應(yīng)用受到極大限制[5]。 來源于自體骨髓組織或脂肪組織的間充質(zhì)干細(xì)胞,取材方便,通過體外分離、培養(yǎng)及富集,可獲得大量的細(xì)胞,如果能將其誘導(dǎo)成leydig細(xì)胞,則可以同時(shí)解決leydig細(xì)胞移植的來源及免疫排斥兩個(gè)問題。普遍認(rèn)為,性腺及腎上腺等類固醇合成器官從來源于中段中胚層的腎上腺生殖器原基發(fā)育而來[6],而Leydig細(xì)胞來源于胚胎發(fā)育期的中腎胚的間質(zhì)細(xì)胞,并認(rèn)為位于管周的成纖維樣細(xì)胞為leydig干細(xì)胞或前體細(xì)胞,leydig細(xì)胞由這些細(xì)胞分化而來[7]。間充質(zhì)干細(xì)胞(mesenchymal Stem Cells,MSCs)與leydig干細(xì)胞都來源于中胚層,因而具有向Leydig細(xì)胞轉(zhuǎn)化的理論可能[8]。普遍認(rèn)為類固醇因子-1(steroidogenic factor 1, SF-1)是睪丸及腎上腺發(fā)育及類固醇激素合成的關(guān)鍵的核內(nèi)受體,在它的調(diào)控下,類固醇合成酶的各個(gè)基因得以表達(dá)[6]。Tomoko Tanaka在2006年用骨髓基質(zhì)細(xì)胞注入發(fā)育期的大鼠睪丸內(nèi),這些細(xì)胞表現(xiàn)出與leydig細(xì)胞相似的特性;同時(shí)將攜帶綠色熒光蛋白的P450scc用腺病毒攜帶AD/4Bp基因后轉(zhuǎn)染體外分離小鼠BMSCs,并在加入cAMP培養(yǎng)液中培養(yǎng)2周后,這些細(xì)胞表現(xiàn)出leydig細(xì)胞的特性,并能分泌睪酮[8]。Gondo等在2008年用這種方法處理BMSCs及脂肪干細(xì)胞(adipose-derived mesenchymal stem cells, AMSCs)后,發(fā)現(xiàn)BMSCs向產(chǎn)生睪酮的leydig細(xì)胞方向分化,而AMSCs則傾向于產(chǎn)生糖皮質(zhì)類激素的腎上腺細(xì)胞分化[6]。Takashi2009年將肝受體同系物-1(liver receptor homolog-1,LRH)以質(zhì)粒轉(zhuǎn)染人的BMSCs及cAMP處理后,這些細(xì)胞表達(dá)腎上腺及睪酮合成的基因[9]。該類實(shí)驗(yàn)表明BMSCs在異位表達(dá)的SF-1的刺激下可以向leydig細(xì)胞方向轉(zhuǎn)化,轉(zhuǎn)錄并表達(dá)睪酮合成過程中所需的各種酶,并具有睪酮分泌功能。該類實(shí)驗(yàn)主要用于研究性腺器官的發(fā)育,由于腺病毒轉(zhuǎn)染的潛在安全問題,誘導(dǎo)后的細(xì)胞在臨床治療中的意義不大,同時(shí)說明在誘導(dǎo)分化為leydig細(xì)胞上BMSCs比ADSCs更適合。 另外的研究主要是利用leydig細(xì)胞在體內(nèi)生長的微環(huán)境在也可將BMSCs誘導(dǎo)向leydig細(xì)胞方向分化。Takashi Yazawa及Tomoko Tanaka等將大鼠BMSCs注射入3周齡發(fā)育期大鼠睪丸,結(jié)果顯示注射的部分BMSCs能向Leydig細(xì)胞分化,并表達(dá)leydig細(xì)胞的各種細(xì)胞標(biāo)志物[8、10]。YanHe Lue等進(jìn)一步探討了BMSCs注射入睪丸的曲精小管及睪丸間質(zhì)的分化結(jié)局,綠色熒光蛋白標(biāo)記的BMSCs分別注射入正常leydig細(xì)胞的大鼠及c-kit基因沉默的W/Wv大鼠(睪丸發(fā)育缺陷,無法形成精原細(xì)胞)睪丸內(nèi),10-12周后注射入正常大鼠的睪丸內(nèi)BMSCs根據(jù)鄰近細(xì)胞的情況,依次分化為與鄰近細(xì)胞相識(shí)的Leydig細(xì)胞、Sertoli細(xì)胞、生殖細(xì)胞系細(xì)胞等體細(xì)胞成分,而注射入睪丸發(fā)育畸形鼠體內(nèi)的BMSCs未發(fā)生分化。雖該然不能完全排除細(xì)胞融合的可能,但是可以說明睪丸各種細(xì)胞生長的微環(huán)境對(duì)移植BMSCs的定向分化具有誘導(dǎo)作用[11]。 上述研究成果顯示BMSCs誘導(dǎo)分化成為Leydig細(xì)胞具有可行性,但距臨床應(yīng)用仍有差距�；蜣D(zhuǎn)染技術(shù)雖然有望在體外大量的誘導(dǎo)BMSCs分化為雄激素分泌細(xì)胞,但該方法必須克服腺病毒轉(zhuǎn)染技術(shù)帶來的生物安全性問題,目前尚屬世界性難題。BMSCs睪丸內(nèi)注射屬實(shí)驗(yàn)性研究,要求受體動(dòng)物在發(fā)育期,對(duì)于Leydig細(xì)胞功能不足或不存在睪丸的受體意義不大,且分化的效率不高,目前通過該途徑很難獲得大量的自體BMSCs分化的Leydig細(xì)胞。然而,以上研究成果提示,雖然目前對(duì)睪丸分化微環(huán)境促進(jìn)BMSCs定向分化的始動(dòng)因素并不明確,但通過體外模擬Leydig細(xì)胞生長的微環(huán)境,在體外實(shí)現(xiàn)BMSCs向Leydig細(xì)胞大量的定向分化具備理論可能性。 綜上所述,針對(duì)Leydig細(xì)胞治療及雄激素分泌組織構(gòu)建研究中關(guān)鍵的種子細(xì)胞來源問題,結(jié)合本課題組前期實(shí)驗(yàn)中,通過差速貼壁法可以大量獲得較純的leydig細(xì)胞的基礎(chǔ)上,將骨髓間充質(zhì)干細(xì)胞與Leydig細(xì)胞共培養(yǎng),利用leydig細(xì)胞生長的微環(huán)境及分泌的可溶性因子誘導(dǎo)BMSCs向Leydig細(xì)胞分化,以解決種子細(xì)胞來源不足的難題。該項(xiàng)研究可為臨床雄性激素缺乏癥提供一條安全有效的途徑,同時(shí)對(duì)于進(jìn)一步研究胚胎發(fā)育過程中l(wèi)eydig干細(xì)胞具體分化過程中的各種細(xì)胞因子的作用及調(diào)控機(jī)制具有借鑒意義。 研究目的 本研究從骨髓基質(zhì)干細(xì)胞開始,通過Ficoll液分離、貼壁、增殖并傳代,富集足夠的BMSCs,并經(jīng)過誘導(dǎo)分化成骨、成脂肪鑒定BMSC的多向分化能力;通過差速貼壁法獲得的大鼠睪丸leydig細(xì)胞的免疫組化分析,證實(shí)差速貼壁法可以獲得較純的大鼠睪丸leydig細(xì)胞;探索共培養(yǎng)體系對(duì)BMSC向leydig細(xì)胞誘導(dǎo)的可能性,并對(duì)誘導(dǎo)后的BMSC進(jìn)行l(wèi)eydig細(xì)胞的各項(xiàng)特異性指標(biāo)進(jìn)行免疫組化、RT-PCR的測(cè)定,檢驗(yàn)誘導(dǎo)的BMSC作為雄激素分泌組織種子細(xì)胞的可行性,為解決組織工程化雄激素分泌組織研究中種子細(xì)胞來源不足的問題提供有效手段。 研究方法 一、密度梯度離心法獲得骨髓基質(zhì)干細(xì)胞及鑒定: 3周齡大鼠的股骨、脛骨,10ml注射器沖洗,1.083g/ml的ficoll去除血細(xì)胞,2天后半量換液,4天后首次全部換液,獲取的貼壁細(xì)胞,并傳代培養(yǎng)。取第三代BMSC進(jìn)行成骨、成脂肪分化誘導(dǎo)。同時(shí)觀察細(xì)胞形態(tài),克隆樣集落形成,并檢測(cè)細(xì)胞增殖情況。 二、差速貼壁法分離純化3周鼠齡大鼠Leydig細(xì)胞:取3周齡雄性Wister大鼠雙側(cè)睪丸,無菌下去除白膜及較大的血管,用膠原酶震蕩消化和400目(Ф33μm)不銹鋼濾網(wǎng)過濾,離心后貼壁培養(yǎng)2小時(shí),棄上清并漂洗3次,加入3%FBS的DMEM/F12培養(yǎng)液培養(yǎng)。 三、共培養(yǎng)體系對(duì)BMSC誘導(dǎo)分化作用:3周齡大鼠睪丸leydig分離后以transwell培養(yǎng)皿雙層間接共培養(yǎng)體系進(jìn)行培養(yǎng),隔1-2天換液,2周、4周后對(duì)誘導(dǎo)的細(xì)胞3β-HSD、LHR免疫化學(xué)染色、免疫熒光染色,PCR檢測(cè)stAR、3β-HSD及LHR的表達(dá)情況及睪酮分泌水平的測(cè)定。 研究結(jié)果 第一部分梯度密度離心法獲得骨髓基質(zhì)干細(xì)胞及鑒定通過剝離股骨、脛骨,10ml注射器沖洗洗骨髓,1.083g/ml的Ficoll液去除紅細(xì)胞,離心后用10%小牛血清的低糖DMEM培養(yǎng)液接種,貼壁4天后首次全量換液,可以獲取的數(shù)量不等的貼壁細(xì)胞,并形成克隆集落樣生長,傳代培養(yǎng)后細(xì)胞形態(tài)、生長能力無明顯變化,3天后即可觀察到可見BMSC呈的克隆集落樣生長,在體外培養(yǎng)條件下可以傳至6-7代。取第三代的BMSC可以誘導(dǎo)向成骨、成脂肪分化,說明獲得的BMSCs具備很強(qiáng)的增殖能力,并多向誘導(dǎo)分化,可用于下一步研究。 第二部分差速貼壁法獲得的LC細(xì)胞成分分析 采用差速貼壁法能夠獲得3周齡大鼠睪丸LC細(xì)胞,經(jīng)3β-HSD、LHR免疫化學(xué)染色分析,結(jié)果顯示在DMEM/F12培養(yǎng)體系內(nèi)培養(yǎng)1天后,幾乎所有的細(xì)胞都表達(dá)3β-HSD、LHR抗體,提示我們?cè)摲椒苡行?duì)leydig細(xì)胞進(jìn)行分離、富集。 第三部分leydig細(xì)胞與BMSCs的共培養(yǎng)體系對(duì)BMSC誘導(dǎo)分化作用的研究通過3周鼠齡的leydig細(xì)胞與BMSCs間接共培養(yǎng)2周、4周后,免疫組化、PCR分析鑒定共培養(yǎng)的BMSCs情況,免疫組化顯示部分BMSC表達(dá)leydig細(xì)胞特異性標(biāo)志物3β-HSD、LHR, RT-PCR反應(yīng)檢測(cè)到leydig細(xì)胞的部分特異性標(biāo)志物stAR、3β-HSD及LHR的mRNA表達(dá),說明在間接共培養(yǎng)條件下BMSC可以向leydig樣細(xì)胞分化。 研究結(jié)論 本研究證實(shí)差速貼壁法能夠有效獲得大鼠睪丸Leydig系細(xì)胞的各級(jí)細(xì)胞成分,通過間接共培養(yǎng)體系,能夠誘導(dǎo)骨髓間質(zhì)干細(xì)胞向leydig樣細(xì)胞方向上分化,并表達(dá)leydig細(xì)胞的特異性標(biāo)志物。我們推測(cè)通過進(jìn)一步分析leydig細(xì)胞分化的始動(dòng)因素及l(fā)eydig細(xì)胞分化過程中的各種所需的各種營養(yǎng)因子,優(yōu)化實(shí)驗(yàn)培養(yǎng)條件將可能誘導(dǎo)BMSCs分化為成熟的leydig細(xì)胞,為臨床上雄性激素缺乏性疾病提供一條理想的治療途徑。
[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 .

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王曉云;邢新;周廣東;劉偉;曹誼林;;共培養(yǎng)大鼠睪丸體細(xì)胞組織工程技術(shù)構(gòu)建雄激素分泌組織[J];中華醫(yī)學(xué)雜志;2007年31期

2 畢宏達(dá);王曉云;周廣東;劉偉;李鳴;邢新;;大鼠睪丸間質(zhì)細(xì)胞體外培養(yǎng)不同時(shí)間3β-羥基類固醇脫氫酶的表達(dá)[J];組織工程與重建外科雜志;2009年01期

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本文編號(hào)：1757658