本文選題：脂肪來源的間充質(zhì)干細胞 + 脫細胞真皮基質(zhì)�。� 參考：《河北醫(yī)科大學》2009年碩士論文

【摘要】： 由于負重關(guān)節(jié)面由透明軟骨組成,軟骨細胞被細胞外基質(zhì)包繞,幾乎沒有遷徙能力,不能聚集到創(chuàng)傷部位,因此,軟骨組織一旦發(fā)生損傷后自我修復(fù)能力十分有限。軟骨缺損常常引起關(guān)節(jié)疼痛及功能障礙甚至殘疾,臨床上缺乏有效的治療方法,從而促進了軟骨組織工程技術(shù)地快速發(fā)展。軟骨組織工程涉及種子細胞、支架材料及生長因子、信號分子和應(yīng)力作用等環(huán)境方面的內(nèi)容。其中種子細胞是影響組織工程學的重要因素。脂肪來源的間充質(zhì)干細胞(adipose-derived mesenchymal stem cells, ADMSCs)由于其強大的增殖和分化能力做為組織工程的種子細胞來源而備受關(guān)注。 目的: 1探討脂肪來源的間充質(zhì)干細胞分離和培養(yǎng)的可行性,為擴大和優(yōu)化軟骨組織工程種子細胞源提供實驗依據(jù)。 2自體脂肪來源的間充質(zhì)干細胞與脫細胞真皮基質(zhì)(acellular derma matrix,ADM)復(fù)合修復(fù)兔膝關(guān)節(jié)軟骨缺損,對修復(fù)組織進行組織學和組織病理學評分,評價修復(fù)效果,為臨床應(yīng)用組織工程學方法修復(fù)關(guān)節(jié)軟骨缺損提供理論依據(jù)和奠定實驗基礎(chǔ)。 方法: 選用健康新西蘭兔36只。剪碎的兔脂肪組織使用0.1%Ⅰ型膠原酶消化離心后獲得自體ADMSCs后進行體外培養(yǎng)。流式細胞儀檢測ADMSCs表面抗原CD44的表達情況。倒置顯微鏡觀察細胞形態(tài)。取原代、第3代ADMSCs,采用細胞計數(shù)法測定細胞生長曲線。 體外特定條件下誘導(dǎo)ADMSCs定向誘導(dǎo)分化為脂肪細胞、成骨細胞及軟骨細胞。ADMSCs成脂方向誘導(dǎo)組成為DMEM,10% FBS,異丁基甲基黃嘌呤(IBMX),200μmol/L地塞米松,10μg/ml胰島素, 1μmol/L吲哚美辛。ADMSCs成骨方向誘導(dǎo)組成為DMEM,10% FBS,0.1μmol/L地塞米松,50μg/mL抗壞血酸,10mmol/Lβ-磷酸甘油。ADMSCs成軟骨誘導(dǎo)組成包括10% FBS、高糖DMEM、10μg/L轉(zhuǎn)化生長因子β1、50μg/ml抗壞血酸、6.25 mg/L胰島素。ADMSCs成脂誘導(dǎo)分化后用油紅O染色,成骨方向誘導(dǎo)分化后用鈷鈣法堿性磷酸酶染色及茜素紅鈣結(jié)節(jié)染色,成軟骨方向誘導(dǎo)分化后進行Ⅱ型膠原免疫組化染色。使用特殊染色來證明ADMSCs的多向分化潛能。 用小牛真皮制備ADM作為細胞載體,ADMSCs體外培養(yǎng)至第3代,制成濃度為5×105/ml的軟骨細胞懸液。將自體ADMSCs與ADM體外復(fù)合培養(yǎng)2d后,行掃描電鏡觀察后,移植于兔膝關(guān)節(jié)軟骨缺損處。 將36只新西蘭兔隨機分為ADMSCs /ADM實驗組、ADM對照組和空白對照組。實驗組髁間窩軟骨缺損處植入ADMSCs /ADM,ADM對照組單純植入脫細胞真皮基質(zhì),空白對照組不作任何植入,分別于術(shù)后6周、9周和12周每組各處死4只動物,取材對修復(fù)組織進行大體、組織學及免疫組化染色觀察,根據(jù)關(guān)節(jié)軟骨組織學評分及組織病理學評分標準對修復(fù)組織進行評分,數(shù)據(jù)輸入SPSS 16.0軟件,用隨機分組的SNK-q法進行統(tǒng)計分析,比較各組的評分差異是否具有統(tǒng)計學意義。 結(jié)果: 1接種24h后可見兔ADMSCs呈長梭形或多邊形貼壁。細胞增殖迅速,原代培養(yǎng)約10～14d左右可以達到80-90%融合。 2 ADMSCs表面抗原的鑒定:流式細胞儀檢測結(jié)果顯示細胞表面抗原CD44呈陽性表達。 3 ADMSCs生長曲線呈“S”形。貼壁2 d后細胞開始增殖, 7 d進入增殖高峰期, 9 d后進入平臺期。 4 ADMSCs成脂誘導(dǎo)分化后經(jīng)油紅染色顯示分泌紅色脂滴的脂肪細胞;成骨誘導(dǎo)分化后堿性磷酸酶染色呈黑色沉淀,茜素紅染色呈鈣化結(jié)節(jié);成軟骨誘導(dǎo)分化后Ⅱ型膠原免疫組化染色陽性。 5電鏡下可見ADM呈多孔疏松結(jié)構(gòu),孔隙率較好,適合ADMSCs生長。 6術(shù)后9周ADMSCs /ADM實驗組大體觀察修復(fù)組織整合較好,無支架材料殘留。ADM對照組和空白對照組為纖維性修復(fù)和無修復(fù)。組織學評分ADMSCs /ADM實驗組與ADM對照組和空白對照組差別有顯著性意義(P0.05), ADM對照組與空白對照組差別無顯著性意義(P0.05);組織病理學評分顯示ADMSCs /ADM實驗組與ADM對照組和空白對照組差別有顯著性(P0.05),ADM對照組與空白對照組無顯著性差異(P0.05)。 7術(shù)后12周ADMSCs /ADM實驗組大體觀察修復(fù)組織表面光滑,與周圍軟骨整合良好。ADM對照組和空白對照組為纖維性修復(fù)和無修復(fù)。組織學評分ADMSCs /ADM實驗組與ADM對照組和空白對照組差別有顯著性(P0.05), ADM對照組與空白對照組差別有顯著性(P0.05);組織病理學評分顯示ADMSCs /ADM實驗組與ADM對照組和空白對照組差別有顯著性(P0.05),ADM對照組與空白組差別有顯著性(P0.05)。 8 II型膠原免疫組化染色顯示ADMSCs /ADM實驗組修復(fù)區(qū)的軟骨細胞含II型膠原,與周圍軟骨結(jié)合良好。 結(jié)論: 1兔脂肪來源的間充質(zhì)干細胞在體外具有生長穩(wěn)定,增殖較快的特點,在體外誘導(dǎo)培養(yǎng)體系下可分化為脂肪細胞、成骨細胞和軟骨細胞,符合間充質(zhì)干細胞多向分化潛能的特征。 2脫細胞真皮基質(zhì)材料適合兔脂肪來源的間充質(zhì)干細胞的黏附和增殖,是軟骨組織工程較為理想的支架,二者復(fù)合后植入軟骨缺損區(qū)能夠修復(fù)關(guān)節(jié)軟骨。 3組織學觀察和Ⅱ型膠原免疫組織化學染色證實脂肪來源的間充質(zhì)干細胞具有向軟骨細胞定向分化的能力。
[Abstract]:Because the articular surface is composed of transparent cartilage, the chondrocytes are wrapped around the extracellular matrix, and there is little migration ability and can not gather to the site of trauma. Therefore, the self repair ability of cartilage tissue is very limited. Cartilage defects often cause joint pain and dysfunction or even disability. There is no effective treatment in clinic. Methods, thus promoting the rapid development of cartilage tissue engineering technology. Cartilage tissue engineering involves seed cells, scaffold materials and growth factors, signal molecules and stress and other environmental aspects. Seed cells are important factors affecting tissue engineering. Mesenchymal stem cells derived from fat (adipose-derived mesenchymal) Stem cells (ADMSCs) has attracted much attention due to its strong proliferation and differentiation ability as a source of seed cells for tissue engineering.
Objective:
1 to explore the feasibility of isolating and culturing adipose derived mesenchymal stem cells, and to provide experimental evidence for expanding and optimizing the seed cell source of cartilage tissue engineering.
2 autologous fat derived mesenchymal stem cells and acellular derma matrix (ADM) were combined to repair the cartilage defects of the knee joint in rabbits. The histological and histopathological scores of the repaired tissues were evaluated and the effect of repair was evaluated. It provided a theoretical basis for the clinical application of tissue engineering method to repair articular cartilage defects and laid the experimental basis for the repair of articular cartilage defects. Foundation.
Method:
36 healthy New Zealand rabbits were selected. The shears rabbit adipose tissue was digested and centrifuged with 0.1% type collagenase. After the autologous ADMSCs was obtained, the expression of the ADMSCs surface antigen CD44 was detected by flow cytometry. The cell morphology was observed by the inverted microscope. The cell growth curve was measured by the cell count method, and the cell count method was used to determine the cell morphology.
Under specific conditions in vitro, ADMSCs was induced to differentiate into adipocytes, and osteoblasts and chondrocytes were induced by.ADMSCs to become DMEM, 10% FBS, isobutyl methylxanthine (IBMX), 200 mol/L dexamethasone, 10 micron g/ml insulin, and 1 u mol/L indomethacin in.ADMSCs osteogenesis induction group as DMEM, 10% FBS, 0.1 muon mol/L ground plug. Amethasone, 50 g/mL ascorbic acid, 10mmol/L beta phosphoric acid glycerol.ADMSCs formed into cartilage induced composition including 10% FBS, high sugar DMEM, 10 micron g/L transforming growth factor beta 1,50 u g/ml ascorbic acid, 6.25 mg/L insulin.ADMSCs in lipid induced differentiation with oil red O staining, after osteogenesis induced differentiation with cobalt calcium alkaline phosphatase staining and alizarin red calcium nodule Immunohistochemical staining of type II collagen was carried out after induction of differentiation in chondrogenic direction. Special staining was used to demonstrate the multipotency of ADMSCs.
ADM was prepared by calf dermis as a cell carrier. ADMSCs was cultured to third generations in vitro, and a chondrocyte suspension with a concentration of 5 x 105/ml was prepared. The autologous ADMSCs was cultured with ADM in vitro for 2D and was observed by scanning electron microscope and transplanted into the cartilage defect of the knee joint of rabbit.
The 36 New Zealand rabbits were randomly divided into the ADMSCs /ADM experimental group, the ADM control group and the blank control group. The experimental group was implanted with ADMSCs /ADM in the intercondylar fossa cartilage defect, and the ADM control group was implanted with the acellular dermal matrix alone. The blank control group had no implantation. 4 animals in each group were killed in each group at 6 weeks, 9 and 12 weeks respectively. On the basis of histological and immunohistochemical staining, the repair tissue was scored on the basis of articular cartilage histology score and histopathology score. The data were entered into SPSS 16 software, and the random group SNK-q method was used for statistical analysis to compare the statistical significance of the scores of each group.
Result:
1 after inoculation with 24h, rabbit ADMSCs showed long spindle shape or polygonal adherence. Cell proliferation was rapid and primary culture was about 10 ~ 14d, which could achieve 80-90% fusion.
2 the identification of ADMSCs surface antigen: flow cytometry results showed that CD44 was positive.
3 the growth curve of ADMSCs showed a "S" shape. After adherence to 2 D, the cells began to proliferate, 7 d entered the peak of proliferation, and entered the plateau stage after 9 d.
4 ADMSCs lipid induced adipocytes were stained by oil red staining and red fat droplets were displayed. After osteogenesis, alkaline phosphatase staining showed black precipitation, alizarin red staining showed calcified nodules, and type II collagen immunohistochemical staining was positive after induction of differentiation.
5 under electron microscope, ADM is porous and porous with good porosity. It is suitable for ADMSCs growth.
6, 9 weeks after the operation, ADMSCs /ADM experimental group was better to observe the integration of repair tissue, no scaffold residual.ADM control group and blank control group were fibrous repair and no repair. Histology score of ADMSCs /ADM experimental group was significantly different from that of ADM control group and blank control group (P0.05), ADM control group and blank control group had no significant difference. Sexual significance (P0.05); histopathology score showed that the difference between the ADMSCs /ADM experimental group and the ADM control group and the blank control group was significant (P0.05), and there was no significant difference between the ADM control group and the blank control group (P0.05).
7, 12 weeks after the operation, ADMSCs /ADM experimental group observed the smooth surface of the repaired tissue, the combination with the surrounding cartilage and the.ADM control group and the blank control group were fibrous repair and no repair. The histological score of the ADMSCs /ADM group was significantly different from that of the ADM control group and the blank control group (P0.05), and the difference between the ADM control group and the blank control group was significant. The histopathology score showed that the difference between the ADMSCs /ADM experimental group and the ADM control group and the blank control group was significant (P0.05), and there was a significant difference between the ADM control group and the blank group (P0.05).
8 immunohistochemical staining of type II collagen showed that the chondrocytes in the repair area of ADMSCs /ADM group contained type II collagen, which was well combined with the surrounding cartilage.
Conclusion:
1 rabbit adipose derived mesenchymal stem cells have the characteristics of stable growth and rapid proliferation in vitro, which can differentiate into adipocytes, osteoblasts and chondrocytes under the in vitro culture system, which conforms to the multi differentiation potential of mesenchymal stem cells.
2 acellular dermal matrix material is suitable for the adhesion and proliferation of mesenchymal stem cells derived from rabbit fat source. It is an ideal scaffold for cartilage tissue engineering. The two composite implanted cartilage defect area can repair articular cartilage.
3 histological observation and type II collagen immunohistochemistry confirmed that adipose derived mesenchymal stem cells had the ability to differentiate into chondrocytes.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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