【摘要】：目的:通過改進(jìn)培養(yǎng)條件建立一個(gè)穩(wěn)定、高效的小鼠生精細(xì)胞體外培養(yǎng)方法,獲得更多的單倍體圓形和長(zhǎng)形精子細(xì)胞。方法:采用兩步酶消化法和差速貼壁法相結(jié)合的方法分離生精細(xì)胞,以曲細(xì)精管來源細(xì)胞為飼養(yǎng)層(MSF),在添加睪酮(T)、卵泡刺激素(FSH)、低濃度(10ng/ml)膠質(zhì)細(xì)胞神經(jīng)營(yíng)養(yǎng)因子(GDNF)和表皮生長(zhǎng)因子(EGF)的DMEM/ F-12培養(yǎng)基中培養(yǎng)生精細(xì)胞;采用RT-PCR檢測(cè)培養(yǎng)前、生精細(xì)胞集落形成、出現(xiàn)圓形精子細(xì)胞時(shí)精母細(xì)胞特異性基因P19和單倍體精子細(xì)胞特異性基因TP1 mRNA的表達(dá),判斷生精細(xì)胞增殖和分化情況;同時(shí)采用激光共聚焦顯微鏡(LSCM)進(jìn)行分選細(xì)胞純度檢測(cè)。結(jié)果:以MSF為飼養(yǎng)層,在添加T、FSH、低濃度GDNF和EGF的情況下,培養(yǎng)第7 d可見較多的生精細(xì)胞克隆并可檢測(cè)到P19 mRNA表達(dá),第12 d可見較多的圓形精子細(xì)胞和個(gè)別活動(dòng)精子以及TP1 mRNA的表達(dá);經(jīng)LSCM鑒定,分選的單倍體圓形精子細(xì)胞純度為82%。結(jié)論:本實(shí)驗(yàn)所建立的培養(yǎng)體系是一個(gè)高效、穩(wěn)定的生精細(xì)胞體外培養(yǎng)體系;激光共聚焦顯微鏡可用于初步鑒定單倍體精子細(xì)胞。 目的:通過對(duì)體外培養(yǎng)獲取的小鼠單倍體精子細(xì)胞印跡基因H19 ICR(Imprinting Control Region)和IGF2r DMR2(Differentially Methylated Region 2)區(qū)甲基化狀態(tài)的檢測(cè),初步評(píng)估體外培養(yǎng)對(duì)生精細(xì)胞印跡基因甲基化狀態(tài)的影響。方法:采用亞硫酸氫鹽測(cè)序(BSP)技術(shù),以印跡基因H19 ICR和IGF2r DMR2區(qū)為擴(kuò)增的目的區(qū)域,對(duì)經(jīng)體外培養(yǎng)獲得的單倍體精子細(xì)胞進(jìn)行甲基化修飾后巢式和半巢式PCR擴(kuò)增,采用DNAman軟件對(duì)特異性擴(kuò)增產(chǎn)物進(jìn)行測(cè)序分析,與Gene Bank上相對(duì)應(yīng)的序列進(jìn)行比對(duì),判斷其甲基化狀態(tài),并與小鼠附睪內(nèi)成熟精子進(jìn)行比較。結(jié)果:小鼠成熟精子中H19 ICR區(qū)15個(gè)CpG位點(diǎn)呈高度甲基化狀態(tài)(96.67%),IGF2r DMR2區(qū)呈非甲基化狀態(tài)(94.29%);而經(jīng)體外培養(yǎng)獲得的單倍體精子細(xì)胞H19 ICR區(qū)甲基化程度顯著降低(69.33%,P0.01),IGF2r DMR2區(qū)甲基化程度顯著增加(44.29%,P0.01)。結(jié)論:昆明白小鼠成熟精子中H19 ICR區(qū)呈高度甲基化狀態(tài);IGF2r DMR2區(qū)呈非甲基化狀態(tài);體外培養(yǎng)產(chǎn)生的單倍體圓形和長(zhǎng)形精子細(xì)胞中,H19 ICR區(qū)發(fā)生了廣泛的甲基化丟失,IGF2r DMR2區(qū)發(fā)生了異常甲基化。
[Abstract]:Aim: to establish a stable and efficient method for in vitro culture of mouse spermatogenic cells by improving the culture conditions, and to obtain more haploid round and long spermatogenic cells. Methods: spermatogenic cells were isolated by two-step enzyme digestion and differential adherent method. (MSF), was used as feeder layer to add testosterone (T), follicular stimulating hormone (FSH),. Spermatogenic cells were cultured in DMEM/ F 鈮，

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