【摘要】：目的 用全骨髓法和密度梯度離心兩種方法對(duì)兔骨髓間質(zhì)干細(xì)胞(MSCs)進(jìn)行體外分離、培養(yǎng)及擴(kuò)增,對(duì)比在培養(yǎng)基中只加入TGF-β_1一種誘導(dǎo)物質(zhì)情況下,兩種方法得到的MSCs的成軟骨能力及差別,旨在前人研究的基礎(chǔ)上,建立一個(gè)更簡(jiǎn)易、實(shí)用的體外誘導(dǎo)培養(yǎng)體系,為軟骨缺損修復(fù)提供更多的實(shí)驗(yàn)依據(jù)。 實(shí)驗(yàn)方法 用3%戊巴比妥鈉1mg/kg耳緣靜脈麻醉,無(wú)菌條件下用骨穿針從兩側(cè)股骨大轉(zhuǎn)子處穿入股骨髓腔內(nèi),骨穿針接10ml注射器(內(nèi)含3000U/ml的肝素0.4ml),每側(cè)各抽取骨髓3～4ml,混合后等分為兩份,一份(A組全骨髓法)中直接加入DMEM-LG培養(yǎng)液,用全骨髓法培養(yǎng)細(xì)胞,另一份(B組密度梯度離心法)加入裝有Percoll分離液的無(wú)菌離心管中,1000r/min離心10min,吸取液面交界處云霧狀單核細(xì)胞層細(xì)胞,制成單細(xì)胞懸液,兩組的第3代細(xì)胞均用轉(zhuǎn)化生長(zhǎng)因子β_1(TGF-β_1)誘導(dǎo)BMSCs向軟骨方向分化。倒置顯微鏡觀察BMSCs的生長(zhǎng)情況,用免疫組織化學(xué)、原位雜交方法檢測(cè)TGF-β_1誘導(dǎo)的細(xì)胞Ⅱ型膠原表達(dá)情況。 實(shí)驗(yàn)結(jié)果 1.相差顯微鏡觀察: 1.1 全骨髓培養(yǎng)組 24h后→部分細(xì)胞貼附在瓶壁上,可見(jiàn)一些單核、長(zhǎng)梭或多角型細(xì)胞
[Abstract]:Objective to isolate, culture and amplify (MSCs) from rabbit bone marrow mesenchymal stem cells by whole bone marrow method and density gradient centrifugation in vitro. Under the condition that only TGF- 尾 _ 1 was added into the medium, the chondrogenic ability and difference of MSCs obtained by the two methods were compared. The purpose of this study was to establish a more simple and practical culture system of MSCs induced in vitro on the basis of previous studies. To provide more experimental evidence for cartilage defect repair. Methods 3% pentobarbital sodium (1mg/kg) was used to anesthetize the auricular margin of rats. Under aseptic condition, the femoral medullary cavity was inserted with bone piercing needle from the bilateral trochanter of femur. 10ml syringe (heparin 0.4ml containing 3000U/ml) was connected with bone puncture needle. Each side of bone marrow was extracted from 3ml of bone marrow. After mixing, the bone marrow was divided into two parts. One part (group A, whole bone marrow method) was directly added into DMEM-LG culture medium, and the cells were cultured by whole bone marrow method. The other (group B density gradient centrifugation) was added to the aseptic centrifuge tube containing the Percoll isolate. The 1000r/min was centrifuged for 10 mins, and the monocyte layer cells in the cloud shape at the junction of the liquid level were absorbed, and the monocytes were made into a single cell suspension. Transforming growth factor 尾 _ 1 (TGF- 尾 _ 1) was used to induce the differentiation of BMSCs into cartilage. The growth of BMSCs was observed by inverted microscope, and the expression of type 鈪，

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