發(fā)布時(shí)間：2018-09-13 15:37

【摘要】：研究背景：骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells, MSCs)作為骨髓中一類非造血類干細(xì)胞,具有自我更新和多向分化潛能,最近發(fā)現(xiàn)還具有低免疫原性的特點(diǎn),因此受到臨床醫(yī)學(xué)研究領(lǐng)域的廣泛關(guān)注。目前,MSCs已被廣泛應(yīng)用于研究和治療各種缺血或損傷性疾病以及器官移植抗免疫排斥反應(yīng)等,并取得了初步效果,干細(xì)胞替代治療有望成為臨床上一種新的治療手段。 乳腺癌術(shù)后患側(cè)上肢淋巴水腫是一種慢性進(jìn)行性疾病,主要原因是由于在腋窩淋巴結(jié)切除或前哨淋巴結(jié)活檢中損傷了引流上肢的淋巴管,導(dǎo)致淋巴回流不暢,淋巴液淤積于皮下,病情晚期上肢腫脹,功能喪失,給患者造成巨大的痛苦,嚴(yán)重影響患者生存質(zhì)量。治療上目前以保守治療為主,但是效果不令人滿意,促進(jìn)淋巴管的再生被認(rèn)為是治療淋巴水腫最直接和有效的方法,本文設(shè)想能不能找到一種方法促進(jìn)淋巴管生成來治療乳腺癌術(shù)后上肢淋巴水腫。 目的：從成人骨髓中分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞并在體外分化誘導(dǎo)成淋巴管內(nèi)皮細(xì)胞。檢測(cè)分化的淋巴管內(nèi)皮細(xì)胞在體外形成淋巴管的功能。 方法：在髂后上棘采集健康成人骨髓,采用直接貼壁法分離成人骨髓間充質(zhì)干細(xì)胞,用L-DMEM培養(yǎng)基培養(yǎng)擴(kuò)增至第3-4代,應(yīng)用流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面標(biāo)志CD90、CD105、CD14、CD34、HLA-DR的表達(dá)率。把細(xì)胞分成兩組,一組用EGM-2培養(yǎng)基培養(yǎng)并加入VEGFC-C(156s),另一組只用EGM-2培養(yǎng)基培養(yǎng),培養(yǎng)7天后,在相差顯微鏡下觀察細(xì)胞的形態(tài)變化,應(yīng)用流式細(xì)胞術(shù)檢測(cè)淋巴管內(nèi)皮細(xì)胞的特異性標(biāo)志物Podoplanin、VEGFR-2和VEGFR-3,并應(yīng)用RT-PCR檢測(cè)相應(yīng)基因的表達(dá)。用細(xì)胞成管實(shí)驗(yàn)分別檢測(cè)實(shí)驗(yàn)組和對(duì)照組淋巴管內(nèi)皮細(xì)胞形成淋巴管的功能。 結(jié)果：貼壁培養(yǎng)的骨髓間充質(zhì)細(xì)胞形態(tài)上為纖維細(xì)胞樣,細(xì)胞培養(yǎng)純化三代后經(jīng)流式細(xì)胞術(shù)測(cè)定細(xì)胞的CD90、CD105的表達(dá)率分別為90.03%和97.86%,而CD14、CD34、HLA-DR的陽性率分別為2.00%、2.18%和1.56%。實(shí)驗(yàn)組經(jīng)EGM-2和VEGF-C(156s)誘導(dǎo)刺激后,分離純化的骨髓間充質(zhì)干細(xì)胞呈鵝卵石樣外觀,Podoplanin、VEGFR-2和VEGFR-3的表達(dá)率分別為26.53%、2.53%和8.55%,而對(duì)照組Podoplanin、VEGFR-2和VEGFR-3的表達(dá)率分別為2.84%、1.61%和2.3%,兩組相比Podoplanin和VEGFR-3具有統(tǒng)計(jì)學(xué)意義(P0.01),兩組間VEGFR-2無統(tǒng)計(jì)學(xué)意義(P0.05)。RT-PCR結(jié)果,試驗(yàn)組可見podoplanin和vegfr-3特異性的條帶,未見vegfr-2的特異性條帶,對(duì)照組只顯示了模糊的podoplanin,而未見vegfr-2和vegfr-3的特異性條帶。細(xì)胞成管實(shí)驗(yàn)顯示誘導(dǎo)后的淋巴管內(nèi)皮細(xì)胞具有初步形成管狀結(jié)構(gòu)的功能。 結(jié)論： 1.成功從成人骨髓中分離培養(yǎng)出骨髓間充質(zhì)干細(xì)胞。 2.分離培養(yǎng)的骨髓間充質(zhì)干細(xì)胞在內(nèi)皮細(xì)胞培養(yǎng)基EGM-2和VEGF-C(156s)的誘導(dǎo)下,可分化為淋巴管內(nèi)皮細(xì)胞。 3.誘導(dǎo)后的淋巴管內(nèi)皮細(xì)胞具有初步形成淋巴管的功能。 4.骨髓間充質(zhì)干細(xì)胞在促進(jìn)淋巴管生成方面有很好的應(yīng)用前景。
[Abstract]:BACKGROUND: Bone marrow mesenchymal stem cells (MSCs), as a kind of non-hematopoietic stem cells in bone marrow, have the potential of self-renewal and multi-directional differentiation. Recently, they have been found to have low immunogenicity, so they have attracted wide attention in the field of clinical medicine. At present, MSCs have been widely used in research and treatment. Various ischemic or injury diseases and organ transplantation have achieved preliminary results, and stem cell replacement therapy is expected to become a new clinical treatment.
Lymphedema of the affected upper extremity after breast cancer surgery is a chronic progressive disease. The main reason is that lymphatic drainage of the upper extremity is impaired during axillary lymph node resection or sentinel lymph node biopsy, resulting in lymphatic reflux obstruction, lymph accumulation in the subcutaneous, late stage of the disease, swelling of the upper extremity, loss of function, causing great pain to the patient. At present, conservative treatment is the main treatment, but the effect is unsatisfactory. Promoting lymphatic regeneration is considered to be the most direct and effective method to treat lymphedema. This article envisages that we can find a way to promote lymphangiogenesis to treat upper limb lymphedema after breast cancer surgery.
AIM: To isolate and culture bone marrow mesenchymal stem cells (BMSCs) from adult bone marrow and differentiate into lymphatic endothelial cells in vitro.
Methods: Adult bone marrow mesenchymal stem cells (BMSCs) were isolated from the posterior superior iliac spine by direct adherence method. The expression of CD90, CD105, CD14, CD34 and HLA-DR on the cell surface was detected by flow cytometry. The cells were divided into two groups. One group was cultured in EGM-2 medium and added with VEGF. C-C (156 s), another group was cultured in EGM-2 medium for 7 days. The morphological changes of the cells were observed under phase contrast microscope. The specific markers of lymphatic endothelial cells, Podoplanin, VEGFR-2 and VEGFR-3, were detected by flow cytometry, and the expression of the corresponding genes was detected by RT-PCR. Lymphatic endothelial cells were used to form lymphatic vessels.
Results: The adherent cultured bone marrow mesenchymal cells were fibroblast-like in morphology. The expression of CD90, CD105 and CD14, CD34, HLA-DR were 90.03%, 97.86% and 2.00%, 2.18% and 1.56% respectively by flow cytometry after three generations of cell culture and purification. After stimulation by EGM-2 and VEGF-C (156 s), the experimental group was isolated and purified. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 were 26.53%, 2.53% and 8.55% respectively. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 in control group were 2.84%, 1.61% and 2.3% respectively. There was no significant difference between the two groups (P The results of RT-PCR showed that there were specific bands of Podoplanin and VEGFR-3 in the experimental group, but no specific bands of VEGFR-2 in the experimental group. The control group showed only vague bands of podoplanin, but no specific bands of VEGFR-2 and VEGFR-3 in the control group.
Conclusion:
1. bone marrow mesenchymal stem cells were successfully isolated from adult bone marrow.
2. Bone marrow mesenchymal stem cells can differentiate into lymphatic endothelial cells induced by EGM-2 and VEGF-C (156 s).
3. the lymphatic endothelial cells after induction have the function of forming lymphatic vessels preliminarily.
4. bone marrow mesenchymal stem cells have a good application prospect in promoting lymphangiogenesis.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 路艷蒙,傅文玉,樸英杰,喬?hào)|訪,安連兵;人骨髓間充質(zhì)干細(xì)胞的超微結(jié)構(gòu)[J];電子顯微學(xué)報(bào);2002年04期

2 梁艷紅;張肇林;田鏵;王長明;王世坤;李鑫;宋濤;;單核細(xì)胞向淋巴管內(nèi)皮細(xì)胞誘導(dǎo)分化的潛能(英文)[J];中國組織工程研究與臨床康復(fù);2010年10期

3 柯金勇;林艷娟;;骨髓間充質(zhì)干細(xì)胞生物學(xué)特性的研究[J];醫(yī)學(xué)綜述;2008年15期

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本文編號(hào)：2241595