本文關(guān)鍵詞： 前列腺素E1 脂肪源性干細(xì)胞 細(xì)胞分離 細(xì)胞增殖　出處：《鄭州大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的 皮下軟組織充填一直是整形外科常見的治療手段,對于體表的皮下軟組織欠豐滿、凹陷、萎縮畸形目前常采用自體游離脂肪或各種人工材料進(jìn)行充填。人工材料因存在排異、創(chuàng)傷大及安全性等問題難以滿足臨床需要,而自體游離脂肪的移植成活率僅為40%～50%,這使得其臨床應(yīng)用受到很大限制。脂肪移植物被吸收的原因之一是成熟脂肪細(xì)胞對缺氧、損傷的耐受力低,且不能增殖更新。而脂肪源性干細(xì)胞(Adipose derived stem cells, ADSCs)則具有強(qiáng)大的增殖分化潛力,對缺氧耐受力更強(qiáng),且自體干細(xì)胞用于移植町避免排異反應(yīng)。更加安全 脂肪源性干細(xì)胞不僅具有跟骨髓間充質(zhì)干細(xì)胞相似的多向分化潛能,而且具有取材方法簡便、來源廣泛、患者創(chuàng)傷小等優(yōu)勢,這都使其成為近年來干細(xì)胞研究領(lǐng)域中的熱點(diǎn)。干細(xì)胞應(yīng)用的一個(gè)重要方向就是脂肪組織工程,由于脂肪干細(xì)胞具有上述優(yōu)點(diǎn),這無疑令脂肪干細(xì)胞成為構(gòu)建工程脂肪組織的種子細(xì)胞的最佳選擇。如何提高脂肪源性干細(xì)胞的增殖分化能力是構(gòu)建工程脂肪組織重要的研究課題。 前列腺素El(prostaglandinEl, PGE1)是血管內(nèi)皮細(xì)胞產(chǎn)生的一種保護(hù)因子,是一種有效的血管擴(kuò)張劑,長期以來主要用于肺動脈高壓的治療,隨著臨床應(yīng)用、研究的進(jìn)展,人們逐漸認(rèn)識到PGE1具有抑制血小板聚集和血栓形成、松弛支氣管平滑肌、保護(hù)缺血性心肌、縮小梗塞面積、抑制巨噬細(xì)胞和白細(xì)胞脫顆粒、改善紅細(xì)胞變形及正性肌力等作用,已廣泛應(yīng)用于冠心病心絞痛、心力衰竭、高脂血癥、糖尿病神經(jīng)病變、慢性腎功能不全等。 已有研究發(fā)現(xiàn)米索前列醇(PGE1類似物)對人前脂肪細(xì)胞的增殖和分化均有較強(qiáng)的刺激作用,而PGEl對ADSCs增殖的作用影響尚未見報(bào)道,研究前列腺素對脂肪源性干細(xì)胞的增殖及分化作用,對探求提高脂肪源性干細(xì)胞移植的成活率具有臨床實(shí)用意義,因此設(shè)計(jì)實(shí)驗(yàn),初步探索PGE1對ADSCs細(xì)胞增殖的作用效果。 方法 用酶消化法提取4例臨床患者腹部皮下脂肪組織中的干細(xì)胞,體外進(jìn)行細(xì)胞培養(yǎng)。對分離得到的脂肪細(xì)胞在顯微鏡下進(jìn)行細(xì)胞形態(tài)觀察,流式細(xì)胞術(shù)檢測細(xì)胞表面標(biāo)志物。當(dāng)脂肪源性干細(xì)胞一次性培養(yǎng)的數(shù)量過多,短期內(nèi)又不會完全使用時(shí),可以將部分凍存起來,以備日后實(shí)驗(yàn)時(shí)再復(fù)蘇使用。用五種不同濃度的PGE1處理培養(yǎng)的細(xì)胞,其中3μmol/L,6μmol/L,9μmol/L和12μmol/L作為實(shí)驗(yàn)組,0μmol/L作為對照組,觀察經(jīng)不同濃度的PGE1處理后細(xì)胞的增殖情況。 結(jié)果 ADSCs培養(yǎng)種植入培養(yǎng)皿48h后有大量梭形細(xì)胞貼壁,呈成纖維細(xì)胞樣生長,胞漿和核仁豐富,細(xì)胞形態(tài)不一,有短突起,呈短梭形或小多角形,細(xì)胞核居中。5d后,細(xì)胞達(dá)到對數(shù)生長期,細(xì)胞成長梭形融合聚集,緊密排列有一定方向性,呈漩渦狀或平行狀排列。9d后細(xì)胞長滿培養(yǎng)瓶,集落彼此融合呈束狀或漩渦狀排列。13d后為典型成纖維細(xì)胞樣生長。流式細(xì)胞術(shù)檢測結(jié)果顯示ADSCs表達(dá)相對特異分子CD44,CD105,CD34,CD45,CDHLA-DR。通過實(shí)驗(yàn)觀察和檢測,證明實(shí)驗(yàn)收獲的細(xì)胞確實(shí)為形態(tài)完整,功能健全的脂肪源性干細(xì)胞。各實(shí)驗(yàn)組的細(xì)胞數(shù)量均有顯著升高,其差異有統(tǒng)計(jì)學(xué)意義(與對照組相比p=0.004),各實(shí)驗(yàn)組內(nèi)相比,部分差異有統(tǒng)計(jì)學(xué)意義(實(shí)驗(yàn)組組內(nèi)相比p0.05)。 結(jié)論 1.實(shí)驗(yàn)中用酶消化法分離培養(yǎng)的細(xì)胞,經(jīng)組織學(xué)及表面標(biāo)志物鑒定確實(shí)為脂肪源性干細(xì)胞； 2.脂肪源性干細(xì)胞經(jīng)不同濃度的PGE1處理后,細(xì)胞數(shù)目呈現(xiàn)遞增趨勢,再根據(jù)統(tǒng)計(jì)結(jié)果,組間差異有統(tǒng)計(jì)學(xué)意義,初步說明了PGE1能夠促進(jìn)脂肪源性干細(xì)胞的增值作用。 3.應(yīng)用統(tǒng)計(jì)軟件進(jìn)行組內(nèi)比較發(fā)現(xiàn)部分實(shí)驗(yàn)組之間差異有統(tǒng)計(jì)學(xué)意義,說明PGE1的濃度達(dá)到一定高度時(shí),細(xì)胞數(shù)量增值的程度與PGE1具有一定的劑量依賴性。
[Abstract]:Background and purpose
The subcutaneous soft tissue filling plastic surgery has been a common treatment for subcutaneous soft tissue skin less fullness, depression, atrophy deformity is often used in autologous free fat or filling all kinds of artificial materials. Due to the existence of artificial materials rejection, trauma and safety problems such as difficult to meet clinical needs, and free autogenous fat graft survival rate of only 40% to 50%, which makes its clinical application is limited. One of the reasons for absorption of fat grafts is mature fat cells to hypoxia, damage tolerance is low, and cannot be updated. The proliferation of adipose derived stem cells (Adipose derived stem cells, ADSCs) has the potential for proliferation and differentiation strong, stronger tolerance to hypoxia, and autologous stem cell transplantation for the town to avoid rejection more secure.
Adipose derived stem cells not only have bone marrow mesenchymal stem cell differentiation of similar, and has drawn a simple method, wide source, small trauma and other advantages, which make it become hot in recent years in the field of stem cell research. The use of stem cells is an important direction for adipose tissue engineering. Because fat stem cells have the advantages, which undoubtedly become the best choice of adipose derived stem cells in adipose tissue engineering seed cells. How to improve the ability of proliferation and differentiation of adipose derived stem cells is an important research topic in the engineering construction of adipose tissue.
Prostaglandin El (prostaglandinEl, PGE1) is a protective factor produced by vascular endothelial cells, is a potent vasodilator, mainly for a long time for the treatment of pulmonary arterial hypertension, with the clinical application, the progress of the research, people gradually realize that PGE1 can inhibit platelet aggregation and thrombosis, relaxation of bronchial smooth muscle, protection ischemic myocardium, reduce infarct size, macrophage and leukocyte degranulation, improve erythrocyte deformability and positive inotropic effect, has been widely used in angina pectoris of coronary heart disease, heart failure, hyperlipidemia, diabetic neuropathy, chronic renal insufficiency.
Studies have found that misoprostol (PGE1 analogue) on human preadipocyte proliferation and differentiation has a strong stimulating effect, while the effect of PGEl on the proliferation of ADSCs has not been reported of prostaglandins on proliferation and differentiation of adipose derived stem cells, to explore the improvement of adipose derived stem cell survival rate has clinical and practical significance transplantation, therefore the design of the experiment, explore the effect of PGE1 on proliferation of ADSCs cells.
Method
The extraction of 4 cases of abdominal subcutaneous adipose tissue in patients with stem cells by enzyme digestion, cells were cultured in vitro. The observation of the isolated adipose cell morphology under the microscope, flow cytometry to detect the surface markers. When adipose derived stem cell number of one-time training too much, but in the short term will not be fully used, can be stored, and then recovery in preparation for days after the experiment. With five different concentrations of PGE1 treatment of cultured cells, including 3 mol/L, 6 mol/L, 9 mol/L and 12 mol/L as experimental group, 0 mol/L as control group, to observe the proliferation of were treated with different concentrations of PGE1 cells.
Result
ADSCs cultivated into a Petri dish of 48h after a large number of fusiform adherent cells were fibroblast like growth, cytoplasm and nucleolus rich cell morphology, short processes, showed short fusiform shape or polygonal small, centrally located nuclei after.5d cells reached the logarithmic growth phase, spindle cell growth fusion aggregation closely arranged in a certain direction, whirlpool or parallel arranged.9d cells covered the culture bottle, the colony of each other were bundle or whorls after.13d for typical fibroblast growth. Flow cytometry showed that the expression of ADSCs specific molecules CD44, CD105, CD34, CD45, CDHLA-DR. by experimental observation and testing, the experimental results proved the harvested cells indeed form a complete, perfect function of adipose derived stem cells. The number of cells in each experimental group were significantly higher, the difference was statistically significant (p=0.004 compared with the control group, the experimental group) The differences were statistically significant (compared with P0.05 in the experimental group).
conclusion
1. in the experiment, the cells were isolated by enzyme digestion, and they were identified as adipose derived stem cells by histology and surface markers.
2., after treated with different concentrations of PGE1, the number of adipose derived stem cells showed an increasing trend. According to the statistical results, there was a statistically significant difference between the groups. It preliminarily indicated that PGE1 could promote the increment of adipose derived stem cells.
3., compared with the statistical software, it was found that the difference between the experimental groups was statistically significant, indicating that the degree of cell number increment has a dose-dependent relationship with PGE1 when the concentration of PGE1 reaches a certain height.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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