本文選題：組織工程 + 種子細胞��； 參考：《山西醫(yī)科大學》2016年碩士論文

【摘要】：背景:關節(jié)軟骨由于無血管、神經(jīng)及淋巴通過,其一旦損傷則很難自行修復,已知組織工程技術促進軟骨的損傷修復,研究證實:在體外,軟骨細胞作為種子細胞在體外培養(yǎng)極易發(fā)生去分化;在體內(nèi),經(jīng)由其發(fā)育而來的修復組織的表型及力學特性均較正常關節(jié)軟骨差,而且隨著體內(nèi)修復時間的推移,修復組織顯現(xiàn)出現(xiàn)快速退變的特征,制約其臨床應用的前景。軟骨單位(chondron)被作為關節(jié)軟骨的基本功能結(jié)構(gòu),其主要由軟骨細胞與細胞周基質(zhì)(PCM)組成,其中PCM對軟骨細胞代謝和力學傳導起著重要作用。體外實驗證實:軟骨單位與軟骨細胞以1:1在海藻酸鈉凝膠球中共培養(yǎng),其生物學特性優(yōu)于其他比例;然而,軟骨單位和1:1共培養(yǎng)組對體內(nèi)軟骨缺損修復效果如何并不是很清楚。目的:探討使用海藻酸鈉立體培養(yǎng)的軟骨單位、立體共培養(yǎng)的軟骨單位與軟骨細胞(1:1)移植對兔膝關節(jié)軟骨缺損修復的作用,并對比分析兩者修復效果。方法:1.選取2月齡新西蘭兔5只,通過酶解法將膝關節(jié)軟骨消化成軟骨細胞和軟骨單位,與1.2%海藻酸鈉凝膠混合,分別制備成密度為5.8×106/mL的軟骨細胞、軟骨單位以及軟骨細胞和軟骨單位(1:1)混懸液,最后使用模具分別制成內(nèi)含單純軟骨細胞、單純軟骨單位和軟骨細胞與軟骨單位(1:1)共培養(yǎng)的三種海藻酸鈉凝膠球(大小約25μL/個);2.選取4月齡新西蘭兔45只(4.0±0.2 Kg),通過取雙側(cè)股骨滑車內(nèi)外側(cè)髁連線中點行直徑為4mm,深度為3mm全層軟骨缺損,隨機分為3組,將軟骨細胞、軟骨單位與軟骨單位和軟骨細胞(1:1)混合體植入缺損處;3.分別于植入后6、12、24周,通過mri評估、大體觀察、he染色、番紅o染色、免疫組化染色、rt-pcr檢測以及tunel原位檢測凋亡狀況,以此來綜合判斷缺損修復情況。結(jié)果:1.mriroberts評分顯示:自6周至24周,三組評分均呈增高趨勢;12周時共培養(yǎng)組較軟骨細胞組和軟骨單位組均明顯增高(p0.05);24周時共培養(yǎng)組較軟骨單位組和軟骨細胞組增高顯著(p0.05)。2.ii型膠原免疫組化:自6周到24周,軟骨單位組和共培養(yǎng)組其表達量呈增多趨勢;在6周和12周軟骨單位組其表達量均高于共培養(yǎng)組,而24周其共培養(yǎng)組高于軟骨單位組。3.wakitani組織修復評分顯示:隨著時間遞增,軟骨單位組及共培養(yǎng)組評分均呈逐漸降低趨勢,且12周與6周相比有統(tǒng)計學意義(p0.05);各時間組共培養(yǎng)組均明顯低于軟骨單位組(p0.05)。4.rt-pcr顯示:6周至24周,共培養(yǎng)組中col-ii,sox-9mrna表達量均逐漸增多,軟骨單位組col-iimrna表達量逐漸增多;6周至12周,軟骨單位組和共培養(yǎng)組col-x,mmp-13mrna表達量呈降低趨勢,24周時兩者均增高,但共培養(yǎng)組較軟骨單位組增高幅度較小;6周和12周軟骨單位組col-ii及sox-9均高于共培養(yǎng)組,而24周兩指標共培養(yǎng)組均高于軟骨單位組;三個時間點內(nèi),col-x及mmp-13共培養(yǎng)組均低于軟骨單位組。5.tunel細胞凋亡檢測顯示:自6周至24周,軟骨細胞組細胞凋亡率呈增高趨勢,且24周較12周增高有統(tǒng)計學意義(p0.05);軟骨單位組細胞凋亡率有增高趨勢;自6周至12周共培養(yǎng)組細胞凋亡率明顯降低(p0.05),而24周較12周共培養(yǎng)組細胞凋亡率增高顯著(p0.05);在三個時間點共培養(yǎng)組細胞凋亡率均低于軟骨單位組和軟骨細胞組。結(jié)論:1.軟骨單位與軟骨細胞(1:1)共培養(yǎng)作為種子細胞,早期對缺損組織有好的修復作用,晚期可延緩修復組織退化。2.軟骨單位具有延緩軟骨細胞凋亡的作用。
[Abstract]:Background: it is difficult to repair the articular cartilage because of no blood vessel, nerve and lymph, and it is difficult to repair itself once the injury is damaged. It is known that tissue engineering technology promotes the repair of cartilage damage. The study confirms that chondrocytes are easily differentiated in vitro as seed cells in vitro; in vivo, the phenotype and force of the repair of tissue through its development in the body. The characteristics of the study were worse than that of the normal articular cartilage, and with the time of repair in the body, the characteristics of rapid degeneration of the repair tissue appeared and the prospect of its clinical application was restricted. The cartilage unit (chondron) was used as the basic functional structure of articular cartilage, which was mainly composed of chondrocytes and cell Zhou Jizhi (PCM), and PCM was used for chondrocytes. Metabolism and mechanical conduction play an important role. In vitro experiments have proved that chondrocytes and chondrocytes are cultured with 1:1 in sodium alginate gel ball, and their biological characteristics are superior to other proportions; however, how the cartilage defect repair effect of cartilage unit and 1:1 co culture group is not very clear. The effects of cartilage unit, co cultured cartilaginous unit and chondrocyte (1:1) transplantation on the repair of articular cartilage defect of the knee in rabbits were studied and compared and analyzed. Methods: 1. 2 month old New Zealand rabbits were selected to digest the articular cartilage of the knee into chondrocytes and cartilaginous units by enzymolysis and mixed with 1.2% sodium alginate gel. The chondrocytes, cartilaginous cells and chondrocytes and cartilage units (1:1) were not prepared with a density of 5.8 x 106/mL. Finally, three kinds of sodium alginate gel balls (size 25 mu L/) co cultured with pure cartilage unit and chondrocyte and cartilage unit (1:1) were made by using molds, respectively; 2. selected 4 month old new West. 45 rabbits (4 + 0.2 Kg) were randomly divided into 3 groups by taking the middle point of the internal and external condyle of the bilateral femur trochlear and the depth of 3mm full layer cartilage defect into 3 groups. The cartilage cells, cartilage units and chondrocytes (1:1) were implanted into the defect. 3. points were compared to 6,12,24 weeks after the implantation, and were evaluated by MRI, gross observation, he staining, O staining, immunohistochemical staining, RT-PCR detection and TUNEL in situ detection of apoptosis were used to determine the defect repair. Results: the scores of the three groups were increased from 6 to 24 weeks, and at 12 weeks, the co culture group was significantly higher than that of the cartilage and cartilage units (P0.05), and the co culture group at 24 weeks. The cartilage unit group and cartilage cell group increased significantly (P0.05).2.ii collagen immunohistochemical staining: the expression of cartilage unit group and co culture group increased from 6 to 24 weeks, and the expression of cartilage unit group in 6 and 12 weeks was higher than that of the co culture group, and the co culture group was higher than the.3.wakitani tissue repair score of the cartilage unit group at the 24 week. With the increase of time, the score of cartilage unit group and co culture group decreased gradually, and the 12 weeks compared with 6 weeks was statistically significant (P0.05). All time group co culture groups were significantly lower than the cartilage unit group (P0.05).4.rt-pcr display: 6 weeks to 24 weeks, the co culture group of col-ii, sox-9mrna expression increased gradually, cartilage unit group col-iimrna The expression amount increased gradually. The expression of col-x and mmp-13mrna in the cartilage unit group and co culture group decreased in 6 weeks to 12 weeks, and both increased at 24 weeks, but the increase in the co culture group was smaller than that of the cartilage unit group. The col-ii and Sox-9 in the cartilage unit group were higher than that of the co culture group at 6 and 12 weeks, and the two index co culture group was higher than the cartilage unit group at the 6 and 12 weeks. Three time points, col-x and MMP-13 co culture group were lower than the cartilage unit group.5.tunel cell apoptosis detection showed that from 6 weeks to 24 weeks, the apoptosis rate of chondrocyte group increased, and 24 weeks higher than 12 weeks (P0.05), apoptosis rate of cartilage unit group increased; from 6 to 12 weeks, Co culture group cell apoptosis The rate of apoptosis was significantly lower (P0.05), while the apoptosis rate of cells in the 24 week group was significantly higher than that in the 12 week group (P0.05). The apoptosis rate in the co culture group at three time points was lower than that of the cartilage unit group and the chondrocyte group. Conclusion: 1. cartilage unit and chondrocyte (1:1) co culture as seed cells, early repair of the defect tissue and late delay can be extended. .2. cartilage units with delayed repair of tissue degradation can retard chondrocyte apoptosis.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R687.4

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