【摘要】：外傷、感染、糖尿病潰瘍以及腫瘤切除術(shù)后等常常造成皮膚和脂肪等軟組織的缺損,軟組織缺損的修復(fù)問(wèn)題長(zhǎng)期困擾著臨床醫(yī)生。目前臨床上的治療手段存在各種各樣的缺點(diǎn),隨著組織工程和干細(xì)胞技術(shù)的發(fā)展與應(yīng)用,利用干細(xì)胞結(jié)合生物材料構(gòu)建皮膚、脂肪等工程化軟組織成為修復(fù)軟組織缺損最有前景的方法。脂肪組織來(lái)源干細(xì)胞是一類來(lái)源于脂肪組織的成體間充質(zhì)干細(xì)胞,具有自我更新和多向分化的能力,并且具有取材容易、取材量大、損傷較小、細(xì)胞增殖快速、干細(xì)胞含量豐富等優(yōu)點(diǎn),是組織工程理想的種子細(xì)胞。本課題首先利用分離的SD大鼠脂肪組織來(lái)源干細(xì)胞,在鼠的皮膚缺損模型中證明其具有促進(jìn)創(chuàng)面愈合的能力,并在此基礎(chǔ)上進(jìn)一步分離獲取人的脂肪組織來(lái)源干細(xì)胞,并與真皮成纖維細(xì)胞混合模擬真皮微環(huán)境,探討人脂肪組織來(lái)源干細(xì)胞在組織工程皮膚制備中的作用;另外將人脂肪組織來(lái)源干細(xì)胞結(jié)合可注射材料明膠微球,制備工程化脂肪組織。通過(guò)基于脂肪組織來(lái)源干細(xì)胞構(gòu)建的組織工程皮膚和脂肪,探索修復(fù)軟組織缺損的新策略。 本課題主要進(jìn)行了以下幾方面的研究: 1大鼠脂肪組織來(lái)源干細(xì)胞在皮膚創(chuàng)面愈合中的應(yīng)用 目的:體外分離培養(yǎng)SD大鼠脂肪組織來(lái)源干細(xì)胞,對(duì)其進(jìn)行生物學(xué)特性研究,并接種于膠原凝膠中構(gòu)建組織工程真皮修復(fù)大鼠皮膚缺損,探討其作為組織工程皮膚種子細(xì)胞的可行性。方法:分離SD大鼠腹股溝部脂肪組織,采用膠原酶消化獲得脂肪組織來(lái)源干細(xì)胞,用MTT法檢測(cè)細(xì)胞的生長(zhǎng)特性,流式細(xì)胞儀檢測(cè)細(xì)胞周期和細(xì)胞表面分子標(biāo)志物,通過(guò)誘導(dǎo)分化檢測(cè)脂肪組織來(lái)源干細(xì)胞的多向分化潛能,在驗(yàn)證獲取的脂肪組織來(lái)源干細(xì)胞是間充質(zhì)干細(xì)胞的基礎(chǔ)上,進(jìn)行Hoechst33342熒光標(biāo)記并與膠原凝膠混合制備組織工程真皮,用于修復(fù)大鼠皮膚缺損,隨機(jī)分為3組:脂肪組織來(lái)源干細(xì)胞復(fù)合膠原移植組(ADSC+Col),單純膠原移植組(Col),空白對(duì)照組(Cont.),并通過(guò)組織學(xué)和愈合速度檢測(cè)修復(fù)效果。結(jié)果:分離培養(yǎng)的脂肪組織來(lái)源干細(xì)胞具有不斷增殖的能力,表達(dá)間充質(zhì)干細(xì)胞的表面標(biāo)志物,具有向脂肪、骨和神經(jīng)等多個(gè)譜系分化的能力;在復(fù)合膠原凝膠修復(fù)皮膚缺損實(shí)驗(yàn)中,脂肪組織來(lái)源干細(xì)胞復(fù)合膠原移植組(ADSC+Col)、單純膠原移植組(Col)、空白對(duì)照組(Cont.)創(chuàng)面愈合時(shí)間分別為:(14.3±1.7),(16.9±2.5)和(21.2±4.2)天,有顯著性差異(P0.05)。組織學(xué)觀察顯示,脂肪組織來(lái)源干細(xì)胞復(fù)合膠原移植組,光鏡下可見(jiàn)大量成纖維細(xì)胞和真皮膠原基質(zhì)長(zhǎng)入創(chuàng)面,同時(shí)上皮覆蓋創(chuàng)面,已形成釘突樣結(jié)構(gòu),但是未見(jiàn)毛囊和汗腺等皮膚附屬器結(jié)構(gòu);單純膠原移植組次之,上皮爬行但未完全覆蓋創(chuàng)面;空白對(duì)照組真皮層中微血管較多,但是上皮爬行速度慢,未覆蓋創(chuàng)面。標(biāo)記Hoechst33342的脂肪組織來(lái)源干細(xì)胞復(fù)合膠原移植2周后,可見(jiàn)藍(lán)色熒光的細(xì)胞核,證明移植的脂肪組織來(lái)源干細(xì)胞存活于創(chuàng)面。結(jié)論:成功分離了大鼠脂肪組織來(lái)源干細(xì)胞,脂肪組織來(lái)源干細(xì)胞復(fù)合膠原構(gòu)建的組織工程真皮,可以修復(fù)皮膚軟組織缺損,促進(jìn)創(chuàng)面愈合,證明脂肪組織來(lái)源干細(xì)胞作為組織工程皮膚構(gòu)建的種子細(xì)胞的可行性。 2人脂肪組織來(lái)源干細(xì)胞在組織工程皮膚制備中的作用 目的:體外分離培養(yǎng)人脂肪組織來(lái)源干細(xì)胞,對(duì)其生物學(xué)特性進(jìn)行研究。間充質(zhì)和上皮的相互作用是體外構(gòu)建組織工程皮膚的基礎(chǔ),目前有關(guān)單一間充質(zhì)細(xì)胞類型在組織工程皮膚制備中對(duì)表皮的形態(tài)發(fā)生研究較多,但是混合間充質(zhì)細(xì)胞對(duì)表皮形態(tài)發(fā)生的作用未見(jiàn)報(bào)道,在獲得人脂肪組織來(lái)源干細(xì)胞基礎(chǔ)上,研究脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體對(duì)表皮形態(tài)發(fā)生的影響。方法:采用膠原酶消化人脂肪組織,低密度接種獲得人脂肪組織來(lái)源干細(xì)胞,流式細(xì)胞儀檢測(cè)細(xì)胞周期和細(xì)胞表面分子標(biāo)志物,通過(guò)誘導(dǎo)分化檢測(cè)人脂肪組織來(lái)源干細(xì)胞的多向分化潛能,在驗(yàn)證獲取的人脂肪組織來(lái)源干細(xì)胞是間充質(zhì)干細(xì)胞的基礎(chǔ)上,進(jìn)一步分離真皮成纖維細(xì)胞和角質(zhì)形成細(xì)胞,MTT法觀察脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體(細(xì)胞數(shù)量比為1:1)的條件培養(yǎng)液對(duì)角質(zhì)形成細(xì)胞增殖能力的影響,并通過(guò)酶聯(lián)免疫吸附試驗(yàn)(ELISA)對(duì)條件培養(yǎng)液成分進(jìn)行檢測(cè),在此基礎(chǔ)上以脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體與膠原凝膠復(fù)合構(gòu)建組織工程真皮,并在其上接種角質(zhì)形成細(xì)胞,組織學(xué)和形態(tài)測(cè)量學(xué)以及透射電子顯微鏡觀察表皮形態(tài)發(fā)生的情況。結(jié)果:分離培養(yǎng)的人脂肪組織來(lái)源干細(xì)胞具有自我更新的能力,表達(dá)間充質(zhì)干細(xì)胞的表面標(biāo)志物,具有向脂肪、骨和軟骨等多個(gè)譜系分化的能力;脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體的條件培養(yǎng)液可以促進(jìn)角質(zhì)形成細(xì)胞的增殖,ELISA檢測(cè)其中富含肝細(xì)胞生長(zhǎng)因子(HGF)和角質(zhì)細(xì)胞生長(zhǎng)因子(KGF也稱為FGF7),在以脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體復(fù)合膠原為真皮構(gòu)建的組織工程皮膚模型中,組織學(xué)分析表明,角質(zhì)形成細(xì)胞在脂肪組織來(lái)源干細(xì)胞與真皮成纖維細(xì)胞的混合體上廣泛增殖,并形成了較厚且分化良好的表皮層結(jié)構(gòu),并在表皮層的棘細(xì)胞層中檢測(cè)到角蛋白10(表皮分化標(biāo)記物)陽(yáng)性,透射電鏡(TEM)觀察可見(jiàn)橋粒和半橋粒樣結(jié)構(gòu),形態(tài)學(xué)測(cè)量所形成的表皮厚度與天然皮膚接近。結(jié)論:成功分離了人脂肪組織來(lái)源干細(xì)胞,其與真皮成纖維細(xì)胞混合有利于雙層組織工程皮膚的構(gòu)建,在較短的時(shí)間內(nèi)構(gòu)建出形態(tài)結(jié)構(gòu)良好類似于天然皮膚的組織工程皮膚,提示脂肪組織來(lái)源干細(xì)胞在組織工程皮膚的構(gòu)建中發(fā)揮著重要的作用。 3基于人脂肪組織來(lái)源干細(xì)胞和可降解明膠微球的工程化脂肪組織構(gòu)建 目的:以EDC交聯(lián)的明膠微球?yàn)橹Ъ?人脂肪組織來(lái)源干細(xì)胞為種子細(xì)胞,生物反應(yīng)器進(jìn)行三維培養(yǎng)并進(jìn)行成脂誘導(dǎo),構(gòu)建工程化的脂肪組織。方法:制備明膠微球,并檢測(cè)其細(xì)胞相容性和組織相容性,將PKH26標(biāo)記的人脂肪組織來(lái)源干細(xì)胞接種于明膠微球上在旋轉(zhuǎn)培養(yǎng)系統(tǒng)(RCCS)中培養(yǎng),觀察細(xì)胞在明膠微球上的生長(zhǎng)狀態(tài),并對(duì)脂肪組織來(lái)源干細(xì)胞與明膠微球復(fù)合體進(jìn)行成脂誘導(dǎo)7天,移植到免疫缺陷小鼠皮下,4周時(shí)取材檢測(cè)細(xì)胞存活情況和脂肪形成。結(jié)果:經(jīng)EDC交聯(lián)的明膠微球均呈圓球體,大小比較均勻,粒徑在75μm-150μm,脂肪組織來(lái)源干細(xì)胞在微球上生長(zhǎng)狀態(tài)良好,植入大鼠皮下,植入部位未見(jiàn)明顯紅腫,傷口無(wú)紅腫、滲液等炎性反應(yīng),組織學(xué)觀察植入明膠微球局部無(wú)明顯炎癥細(xì)胞浸潤(rùn)。旋轉(zhuǎn)培養(yǎng)系統(tǒng)中,脂肪組織來(lái)源干細(xì)胞在明膠微球上吸附均勻,生長(zhǎng)良好,經(jīng)過(guò)體外成脂誘導(dǎo)7天后,在mRNA水平可檢測(cè)到脂肪細(xì)胞相關(guān)基因PPAR-γ和C/EBPα,移植入免疫缺陷小鼠皮下,無(wú)論是對(duì)照組(未誘導(dǎo))還是實(shí)驗(yàn)組(成脂誘導(dǎo))在術(shù)后4周時(shí)都可以檢測(cè)到PKH26標(biāo)記的脂肪組織來(lái)源干細(xì)胞在微球周邊存活,經(jīng)成脂誘導(dǎo)7天的脂肪組織來(lái)源干細(xì)胞和明膠微球聚合物在裸鼠皮下可以形成脂肪樣組織。結(jié)論:明膠是一種具有較好的細(xì)胞親和性、生物相容性和生物可降解性的天然高分子材料,利用明膠制備明膠微球,以脂肪組織來(lái)源干細(xì)胞為種子細(xì)胞,在旋轉(zhuǎn)培養(yǎng)系統(tǒng)中進(jìn)行三維培養(yǎng),可以為臨床使用提供一種工程化脂肪組織填充物。
[Abstract]:Injuries, infections, diabetic ulcers, and tumor resection often cause soft tissue defects such as skin and fat. The problem of repairing soft tissue defects has long plagued clinicians. At present, there are various shortcomings in the clinical treatment methods, with the development and application of tissue engineering and stem cell technology and the use of stem cells. The construction of soft tissue, such as skin and fat, is the most promising way to repair soft tissue defects. The stem cells derived from adipose tissue are adult mesenchymal stem cells derived from adipose tissue, which have the ability of self renewal and multidifferentiation, and are easy to obtain, have large material, small damage, and rapid cell proliferation. The stem cells are the ideal seed cells for tissue engineering. First, we use the isolated SD rat adipose tissue derived stem cells to prove that it has the ability to promote wound healing in the skin defect model of rats, and further separate the stem cells from the human adipose tissue and make fiber from the dermis on this basis. The function of human adipose tissue derived stem cells in the preparation of tissue engineered skin was investigated by the mixed simulation of dermal microenvironment, and the adipose tissue derived stem cells were combined with the injectable gelatin microspheres to prepare the engineered adipose tissue. The tissue engineered skin and fat based on the stem cells derived from adipose tissue were explored to repair the tissue engineering skin and fat. A new strategy for soft tissue defects.
This subject is mainly studied in the following aspects:
Application of 1 rat adipose tissue derived stem cells in skin wound healing
Objective: to isolate and culture the stem cells from the adipose tissue of SD rats in vitro, to study the biological characteristics of the stem cells, and to construct tissue engineering dermis in collagen gel to repair the skin defect of rats, and to explore the feasibility of it as a tissue engineering skin seed cell. Methods: to isolate the groin adipose tissue of the SD rats and obtain the collagenase digestion. The stem cells were derived from the adipose tissue, and the growth characteristics of the cells were detected by MTT. Flow cytometry was used to detect the cell cycle and surface molecular markers. The multidirectional differentiation potential of stem cells derived from adipose tissue was detected by induction of differentiation. On the basis of verifying the derived stem cells of the adipose tissue derived from mesenchymal stem cells, Hoechst33 342 fluorescent markers were mixed with collagen gel to prepare tissue engineering dermis, which was used to repair the skin defect of rats. It was randomly divided into 3 groups: adipose tissue derived stem cell collagen transplantation group (ADSC+Col), simple collagen transplantation group (Col), blank control group (Cont.), and the repair effect through histology and healing speed. Adipose tissue derived stem cells have the ability to proliferate, express the surface markers of mesenchymal stem cells, and have the ability to differentiate into multiple lineages, such as fat, bone and nerve. In the compound collagen gel repair of skin defects, the adipose tissue derived stem cell collagen graft group (ADSC+Col), simple collagen transplantation group (Col), blank The wound healing time of group (Cont.) was (14.3 + 1.7), (16.9 + 2.5) and (21.2 + 4.2) days, with significant difference (P0.05). Histological observation showed that the adipose tissue derived stem cells were combined with collagen transplantation group, and a large number of fibroblasts and dermal collagen matrix were found to grow into the wound surface under light microscope, and the epithelium covered the wound, and the PNP like structure had been formed. But there was no skin appendage structure such as hair follicle and sweat gland, and simple collagen transplantation group, the epithelium crawled but not completely covered the wound, and there were more microvascular in the blank control group, but the speed of the epithelium crawled slowly, and the wound was not covered. The blue fluorescence was visible after 2 weeks after the transplantation of Hoechst33342 fat tissue stem cells combined with collagen transplantation. It was proved that the stem cells from the adipose tissue derived from the transplanted stem cells survived the wound. Conclusion: the stem cells derived from the adipose tissue derived from the adipose tissue were successfully isolated and the tissue engineered dermis constructed by the stem cells of the adipose tissue, which could repair the soft tissue defects of the skin, promote the healing of the wound, and prove that the stem cells derived from the adipose tissue were used as tissue engineering skin. The feasibility of building seed cells in skin.
The role of 2 human adipose tissue derived stem cells in tissue engineering skin preparation
Objective: to isolate and culture human adipose tissue derived stem cells in vitro, and to study their biological characteristics. The interaction of mesenchymal and epithelial cells is the basis for the construction of tissue engineered skin in vitro. At present, there are more studies on the morphogenesis of the epidermis in the preparation of tissue engineering skin, but mixed mesenchymal cells are mixed. The effect of epidermal morphogenesis was not reported. On the basis of obtaining human adipose tissue derived stem cells, the effects of the mixture of adipose tissue derived stem cells and dermal fibroblasts on the morphogenesis of epidermis were studied. Methods: using collagenase to digest human adipose tissue, low density inoculation to obtain human adipose tissue derived stem cells, flow finer Cell cycle and cell surface molecular markers were detected by cytosgraph, and the multidirectional differentiation potential of stem cells derived from human adipose tissue was detected by induction of differentiation. On the basis of verifying the derived stem cells derived from human adipose tissue derived from mesenchymal stem cells, the dermis fibroblasts and keratinocytes were further separated, and adipose tissue was observed by MTT method. The effect of the conditioned medium of source stem cells and dermal fibroblasts (the number of cells to 1:1) on the proliferation of keratinocytes and detection of the composition of the conditioned medium by enzyme linked immunosorbent assay (ELISA), and on this basis, the mixture and collagen of the stem cells and the dermal fibroblasts were derived from the adipose tissue. Gel composite construction of tissue engineering dermis, and inoculating keratinocytes on it, histology and morphometry and transmission electron microscopy to observe the occurrence of epidermal morphogenesis. Results: the isolated human adipose tissue derived stem cells have the ability to renew themselves, to reach the surface markers of mesenchymal stem cells, and to have the direction of lipid. The ability to differentiate multiple lineages, such as fat, bone, and cartilage; the conditioned medium of a mixture of adipose tissue derived stem cells and dermal fibroblasts can promote the proliferation of keratinocytes. ELISA detection is rich in hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF, also known as FGF7), and in the stem cells derived from adipose tissue. The histologic analysis of the tissue engineering skin model constructed by the mixture of the dermal fibroblasts and the complex collagen of the dermal fibroblasts showed that the keratinocytes proliferate extensively on the mixture of adipose tissue derived stem cells and dermis fibroblasts, forming a thicker and well differentiated epidermis and in the spinous layer of the epidermis. Detection of keratin 10 (epidermal differentiation marker) positive, transmission electron microscopy (TEM) observation of the structure of grained and half bridge particles, morphological measurement of the thickness of the epidermis close to the natural skin. Conclusion: the successful separation of human adipose tissue derived stem cells, and its mixed with dermal fibroblast cells is conducive to the construction of double-layer tissue engineering skin. The construction of tissue engineered skin similar to natural skin is constructed in a short period of time, suggesting that adipose tissue derived stem cells play an important role in the construction of tissue engineered skin.
3 construction of engineered adipose tissue based on human adipose tissue derived stem cells and degradable gelatin microspheres
Objective: EDC crosslinked gelatin microspheres were used as scaffolds. Human adipose tissue derived stem cells were seeded cells. The bioreactor was used for three-dimensional culture and lipid induction, and engineered adipose tissue was constructed. Methods: gelatin microspheres were prepared, and their cellular compatibility and tissue compatibility were detected, and PKH26 labeled human adipose tissue derived stem cells were connected. The growth state of the cells on gelatin microspheres was observed on gelatin microspheres, and the growth state of the cells on gelatin microspheres was observed. The fat tissue derived stem cells and gelatin microspheres complex were induced for 7 days. The cells were transplanted subcutaneously into the immune deficient mice. The survival of the cells and the formation of fat were measured at 4 weeks. The results were as follows: gelatin crosslinked by EDC The microspheres were round spheroids, the size was more uniform, the size of the particles was 75 M-150 mu m. The stem cells of the adipose tissue derived from the microspheres were well grown on the microspheres. Adipose tissue derived stem cells were evenly adsorbed on gelatin microspheres and grew well. After 7 days of lipid induction in vitro, adipocyte related genes PPAR- gamma and C/EBP alpha could be detected at the mRNA level. The transplanted cells were transplanted subcutaneously into the immunodeficient mice. Both the control group (uninduced) or the experimental group (fat induced) could detect PKH at the end of the operation. 26 labeled adipose tissue derived stem cells survived the microspheres, and adipose tissue derived stem cells and gelatin microspheres could form adipose tissue subcutaneously in nude mice after 7 days of lipid induction. Conclusion gelatin is a natural polymer material with better cellular affinity, biocompatibility and biodegradability. Gelatin microspheres were prepared with the stem cells derived from adipose tissue as the seed cells, and three dimensional culture was carried out in the rotation culture system. It could provide a kind of Engineering fat tissue filling for clinical use.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells[J];Science in China(Series C:Life Sciences);2007年04期

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本文編號(hào)：2157659