發(fā)布時(shí)間：2018-01-27 03:02

  本文關(guān)鍵詞： 脂肪干細(xì)胞 增殖 成脂化 倍增　出處：《鄭州大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：1目的研究表明在實(shí)驗(yàn)室進(jìn)行體外培養(yǎng)時(shí),人脂肪干細(xì)胞(adipose-derived stem cells,ADSCs)與骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)有類(lèi)似的生物學(xué)特性,即同樣具有強(qiáng)大的自體復(fù)制、跨胚層或跨系分化的能力,在長(zhǎng)期體外培養(yǎng)過(guò)程中仍然保持多向分化的能力,同時(shí)遺傳背景穩(wěn)定,移植后排異反應(yīng)較少等優(yōu)點(diǎn),并且脂肪干細(xì)胞具有取材方便、自體來(lái)源即可滿(mǎn)足需要等優(yōu)點(diǎn),已成為近年來(lái)研究的熱點(diǎn)。目前臨床應(yīng)用中獲取人脂肪干細(xì)胞的方法,主要是將脂肪抽吸術(shù)后得到的脂肪混合液用Ⅰ型膠原酶消化法進(jìn)行分離、培養(yǎng),但在吸脂術(shù)腫脹麻醉液使用過(guò)程中,麻醉藥及藥物濃度對(duì)脂肪細(xì)胞及分離培養(yǎng)得到的脂肪干細(xì)胞的生物學(xué)特性是否存在負(fù)面影響,知之甚少,國(guó)內(nèi)外也少見(jiàn)相關(guān)研究報(bào)道。大鼠的脂肪組織來(lái)源相對(duì)豐富,同時(shí)涉及的倫理問(wèn)題及試驗(yàn)項(xiàng)目限制均較少,是良好的試驗(yàn)材料。本試驗(yàn)在現(xiàn)有基本成熟的人和動(dòng)物體內(nèi)提取脂肪組織并由此培養(yǎng)脂肪干細(xì)胞的操作基礎(chǔ)上,在大鼠身上提取脂肪組織進(jìn)而培養(yǎng)脂肪干細(xì)胞,并探尋不同濃度羅哌卡因?qū)Υ笫笾靖杉?xì)胞的增殖能力及成脂化的影響,對(duì)脂肪干細(xì)胞獲取過(guò)程中麻醉方案的最佳方式提供基礎(chǔ)研究依據(jù),為臨床上高效能獲取脂肪干細(xì)胞,減少細(xì)胞損耗,提高脂肪干細(xì)胞的利用率,并利用其移植修復(fù)皮膚軟組織缺損和在整形外科其他方面的應(yīng)用提供理論依據(jù)。2材料與方法2.1設(shè)計(jì):細(xì)胞學(xué)體外觀察2.2材料:來(lái)源于4周齡大鼠雙側(cè)腹股溝脂肪組織2.3試驗(yàn)方法:脂肪干細(xì)胞的分離培養(yǎng)→麻醉腫脹液干預(yù)→CCK-8試劑盒檢測(cè)脂肪干細(xì)胞增殖能力→成脂能力檢測(cè)2.4觀察指標(biāo)(1)大鼠脂肪干細(xì)胞形態(tài)學(xué)特征(2)脂肪干細(xì)胞的增殖能力(3)細(xì)胞成脂分化率結(jié)果原代培養(yǎng)的細(xì)胞呈較大的類(lèi)球形單個(gè)核細(xì)胞,消化傳代后,細(xì)胞生長(zhǎng)速度明顯較原代細(xì)胞增快,2~3天即可覆蓋約85%以上。CCK-8實(shí)驗(yàn)發(fā)現(xiàn),25μmol/L、50μmol/L、100μmol/L的羅哌卡因處理脂肪干細(xì)胞24h,48h,72h后吸光度值明顯下降,與對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義。實(shí)驗(yàn)說(shuō)明羅哌卡因處理可顯著降低脂肪干細(xì)胞的增殖能力,在24h時(shí),濃度越高,抑制干細(xì)胞增值能力越強(qiáng),在48h和72h,濃度高低與抑制作用無(wú)明顯差別。對(duì)照組脂肪干細(xì)胞成脂分化比例為(44.61±4.43)%,25μmol/L羅哌卡因處理組為(46.30±3.25)%,50μmol/L羅哌卡因處理組為(49.46±2.34)%,100μmol/L羅哌卡因處理組為(50.07±5.29)%,實(shí)驗(yàn)說(shuō)明低濃度羅哌卡因處理后對(duì)脂肪干細(xì)胞的成脂化能力無(wú)明顯影響,高濃度羅哌卡因處理后對(duì)脂肪干細(xì)胞的成脂化能力存在影響。當(dāng)濃度上升至一定數(shù)值時(shí),脂肪干細(xì)胞成脂化率不隨濃度增加而改變,但仍需進(jìn)一步試驗(yàn)證實(shí)。結(jié)論羅哌卡因處理可顯著降低脂肪干細(xì)胞的增殖能力,在24h時(shí),濃度越高,抑制干細(xì)胞增值能力越強(qiáng),在48h和72h,濃度高低與抑制作用無(wú)明顯差別。低濃度羅哌卡因處理后對(duì)脂肪干細(xì)胞的成脂化能力無(wú)明顯影響,高濃度羅哌卡因處理后對(duì)脂肪干細(xì)胞的成脂化能力存在影響。當(dāng)濃度上升至一定數(shù)值時(shí),脂肪干細(xì)胞成脂化率不隨濃度增加而改變,但仍需進(jìn)一步試驗(yàn)證實(shí)。
[Abstract]:Objective to study the 1 that were cultured in the laboratory, human adipose derived stem cells (adipose-derived stem cells, ADSCs) and bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) have similar biological properties, which also has the powerful ability of self replication, cross germ layer or transdifferentiation, in the long term in vitro culture still retain the capacity for multilineage differentiation process, and stable genetic background, rejection of the advantages of less, and adipose stem cells have convenient, autologous can satisfy the needs and other advantages, has become a research hotspot in recent years. The current clinical application of human adipose derived stem cells access method, which is used primarily by type collagenase digestion fat mixture obtained after liposuction culture were isolated, but in the process of using liposuction tumescent fluid, drug and drug concentration on fat Whether or not the culture has negative impact on the biological characteristics of adipose derived stem cells and isolated, poorly understood, at home and abroad are seldom reported. Relatively abundant adipose tissue derived from rat, and relates to the project problems and test ethical limits are less, is a good test material in this experiment. The existing basic mature man and in vivo animal adipose tissue and adipose derived stem cells on the basis of operational training, in rats from the adipose tissue and cultured stem cells, and explore the proliferation capacity of different concentrations of ropivacaine on rat adipose derived stem cells and the effects of fat, and provide basic evidence for the best way of anesthesia scheme fat stem cells in the process of acquiring the clinical effective acquisition of adipose derived stem cells, reduce the cell loss, improve the utilization rate of adipose derived stem cells, and the transplantation for repairing skin and soft Provide a theoretical basis for the design of.2 materials and methods 2.1 tissue defect and other aspects of the application in plastic surgery: the cytology in vitro observation of 2.2 materials: from bilateral inguinal adipose tissue of 4 week old rats 2.3 test methods: culture, anesthetic liquid intervention CCK-8 kit, adipose stem cell proliferation and adipogenic ability to detect 2.4 observation the separation index of adipose derived stem cells (1) cell morphological characteristics of rat adipose derived stem (2) adipose derived stem cells (3) cell proliferation and adipogenic differentiation rate of the cultured cells were spherical single nuclear cells large, after digestion and passage, the cell growth rate was significantly faster than primary cells, 2~3 days covered above about 85%.CCK-8 experiment, 25 mol/L, 50 mol/L, 100 mol/L ropivacaine treatment of adipose derived stem cells 24h, 48h, 72h after the absorbance value decreased significantly, compared with the control group, there was statistically significant difference Yi. Experiment shows that ropivacaine treatment can significantly reduce the fat stem cell proliferation in the 24h, the higher the concentration, inhibition of stem cells proliferation is stronger in 48h and 72h, concentration and inhibition of no significant difference. Group of adipose derived stem cells into fat differentiation ratio as control (44.61 + 4.43)%, 25 mol/L ropivacaine treatment group (46.30 + 3.25)%, 50 mol/L ropivacaine group was (49.46 + 2.34)%, 100 mol/L ropivacaine group was (50.07 + 5.29)%. The experiment shows that low concentration of ropivacaine after treatment of adipogenic ability has no obvious effect on adipose derived stem cells, influence on fat the adipogenic ability of stem cells in the presence of high concentrations of ropivacaine after treatment. When the concentration increased to a certain value, and the change of adipose derived stem cells into fat rate with the increase of concentration, but still need to be further confirmed. Conclusion ropivacaine treatment can significantly reduce the fat stem cells The ability of proliferation in the 24h, the higher the concentration, inhibition of stem cells proliferation is stronger in 48h and 72h, concentration and inhibition of no significant difference. After treatment with low concentration of ropivacaine on adipogenic ability has no obvious effect on adipose derived stem cells, adipose stem cells effects on adipogenic ability of high concentration ropivacaine after treatment. When the concentration increased to a certain value, and the change of adipose derived stem cells into fat rate with the increase of concentration, but still need to be further confirmed.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R622

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