本文選題：分層軟骨 + 分層軟骨細(xì)胞�。� 參考：《大連醫(yī)科大學(xué)》2017年博士論文

【摘要】：研究背景:正常的關(guān)節(jié)軟骨是一種不均一的分層組織,這種分層結(jié)構(gòu)對維持關(guān)節(jié)軟骨的結(jié)構(gòu)和功能至關(guān)重要。自體軟骨移植被認(rèn)為是治療軟骨缺損最常用而且最有效的手段。但是,自體軟骨細(xì)胞移植并未將關(guān)節(jié)軟骨不同層之間的天然差異加入考慮。最近越來越多的研究著重于再生分層軟骨,而不只是再生均一一致的軟骨組織。使用不同層的軟骨細(xì)胞再生相應(yīng)的分層是其中一種手段之一。不同層的軟骨細(xì)胞能夠通過組織學(xué)水平分離,并且能夠在體外維持一定的分層特性。在體外研究中發(fā)現(xiàn)使用不同層軟骨細(xì)胞分別再生相應(yīng)層的軟骨組織能夠維持不同層相應(yīng)的特性。但是不同層的軟骨細(xì)胞在體外短暫單層培養(yǎng)擴(kuò)增就失去其原有的分層特性,而且很難再次重新獲得這種分層特性。那些使用不同層軟骨細(xì)胞修復(fù)軟骨損傷的研究,均采用低代擴(kuò)增或者未擴(kuò)增的不同層軟骨細(xì)胞。然而,臨床上原代軟骨細(xì)胞必須通過幾代擴(kuò)增后才能獲得足夠的數(shù)量。因此,需要一種方法能夠使用較少的短暫擴(kuò)增或者未擴(kuò)增的不同層軟骨細(xì)胞來再生分層軟骨。軟骨細(xì)胞和間充質(zhì)干細(xì)胞共培養(yǎng)修復(fù)軟骨損傷,被證明是一種有望代替單純軟骨細(xì)胞修復(fù)軟骨損傷的手段。從關(guān)節(jié)非負(fù)重區(qū)獲得的低代擴(kuò)增或者未擴(kuò)增的軟骨細(xì)胞與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)被證明能夠產(chǎn)生與單獨(dú)軟骨細(xì)胞成軟骨相似的效果。因此,這種方法可以減少軟骨細(xì)胞的需求數(shù)量,這樣就可以使用短暫擴(kuò)增甚至不擴(kuò)增的軟骨細(xì)胞用來修復(fù)軟骨損傷,而這些細(xì)胞由于沒有去分化過程,所以仍維持分層特性。此外,使用共培養(yǎng)技術(shù)時(shí),由于可能不需要軟骨細(xì)胞擴(kuò)增,所以軟骨損傷修復(fù)甚至可以變成一步操作。但是,不同層的軟骨細(xì)胞與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)后,兩者是否能夠一起維持不同層的特性仍然不明確。在這篇研究中,我們使用一種常用的體外三維培養(yǎng)體系,海藻酸鈉水凝膠體外培養(yǎng)體系,用來研究不同層的軟骨細(xì)胞和骨髓間充質(zhì)干細(xì)胞共培養(yǎng)后,在體內(nèi)外是否仍能夠維持其不同層的特性。第一部分不同層軟骨細(xì)胞的分離和體外單層培養(yǎng)環(huán)境下分層特性的維持目的:1.提取表層和中深層軟骨細(xì)胞;2.驗(yàn)證原代表層和中深層軟骨細(xì)胞之間的基因表達(dá)、蛋白合成和增殖速度的差異;3.檢測傳代后的表層和中深層軟骨細(xì)胞之間基因表達(dá)、蛋白合成和增殖速度的差異;4.分離并驗(yàn)證骨髓間充質(zhì)干細(xì)胞。方法:使用顯微取皮刀提取1月齡新西蘭兔膝關(guān)節(jié)股骨面表層約100um厚的軟骨組織作為表層軟骨細(xì)胞來源,剩下的軟骨組織作為中深層軟骨細(xì)胞來源。通過二型膠原酶消化獲得原代的表層和中深層軟骨細(xì)胞。抽取1月齡新西蘭兔股骨及脛骨內(nèi)骨髓,利用骨髓間充質(zhì)干細(xì)胞貼壁的特性分離獲得骨髓間充質(zhì)干細(xì)胞。貼壁的骨髓間充質(zhì)干細(xì)胞通過多向誘導(dǎo)證明其多項(xiàng)分化能力。對原代表層和中深層軟骨細(xì)胞(p0代)進(jìn)行col2a、acan和prg4基因檢測,以gapdh作為內(nèi)參,使用2-ΔΔct方法進(jìn)行相對比較。并且將原代表層和中深層軟骨細(xì)胞加到96孔板中進(jìn)行單層培養(yǎng)細(xì)胞增殖實(shí)驗(yàn)。第3、5、7、10天,使用cck-8(cellcountingkit-8)檢測增殖率。原代表層和中深層軟骨細(xì)胞長至90%融合時(shí)定義為p1代,以此類推,對p1和p2代同樣進(jìn)行基因和蛋白質(zhì)水平的檢測,并進(jìn)行增殖速度的檢測。結(jié)果:發(fā)現(xiàn)原代表層和中深層軟骨細(xì)胞之間存在明顯差異,即表層軟骨細(xì)胞表達(dá)更高的prg4基因和col2a基因,中深層軟骨細(xì)胞表達(dá)更高的acan基因。體外單層培養(yǎng)增殖實(shí)驗(yàn)發(fā)現(xiàn),中深層軟骨細(xì)胞增殖速度更快。然而體外擴(kuò)增一代后,增殖速度差異消失,而基因表達(dá)和蛋白水平的差異逐漸縮小,p2代時(shí)差異消失。抽取的骨髓貼壁的細(xì)胞呈現(xiàn)成纖維細(xì)胞樣。而擴(kuò)增后到第三代的骨髓間充質(zhì)干細(xì)胞能夠被誘導(dǎo)向脂肪細(xì)胞、成骨細(xì)胞和軟骨細(xì)胞方向。結(jié)論:1.通過組織學(xué)分離的p0代表層軟骨細(xì)胞和中深層軟骨細(xì)胞呈現(xiàn)不同的特性;2.隨著傳代,不同層軟骨細(xì)胞的基因表達(dá)、蛋白水平和增殖速度差異逐漸消失。第二部分不同層軟骨細(xì)胞與骨髓間充質(zhì)干細(xì)胞體外三維共培養(yǎng)環(huán)境下細(xì)胞外基質(zhì)成分的變化目的:1.體外使用海藻酸鈉水凝膠三維培養(yǎng)體系將表層和中深層軟骨細(xì)胞分別與骨髓間充質(zhì)干細(xì)胞共培養(yǎng);2.檢測不同時(shí)間點(diǎn)(第1、7、21天)時(shí)軟骨細(xì)胞外基質(zhì)成分的基因表達(dá)情況;3.檢測不同時(shí)間點(diǎn)(第1、7、21天)時(shí)軟骨細(xì)胞外基質(zhì)主要成分(gag)含量、dna含量變化以及l(fā)ubricin分泌情況。方法:將p0代表層或中深層軟骨細(xì)胞單獨(dú)或與骨髓間充質(zhì)干細(xì)胞以1:2比例混合后,按6×106/ml細(xì)胞濃度和2%海藻酸鈉混合,使用注射器一滴一滴將海藻酸鈉水凝膠滴入到凝固液(102mm氯化鈣)中形成凝固的小珠,加入到成軟骨誘導(dǎo)培養(yǎng)基中培養(yǎng)。于第1、7、21天時(shí)收獲小珠。分別檢測基因表達(dá)情況和gag、dna含量。取培養(yǎng)第1、7、21天的培養(yǎng)24小時(shí)的上清用elisa法測量lubricin濃度。結(jié)果:基因表達(dá)方面第1天和第7天時(shí),中深層軟骨細(xì)胞共培養(yǎng)組相比于表層軟骨細(xì)胞共培養(yǎng)組表達(dá)更高的acan基因,更低的prg4基因,而col2a水平無明顯差異。不同層軟骨細(xì)胞組之間也有同樣的趨勢。培養(yǎng)第7天時(shí)中深層軟骨細(xì)胞共培養(yǎng)組和中深層軟骨細(xì)胞組分別比表層軟骨細(xì)胞共培養(yǎng)組和表層軟骨細(xì)胞組沉積更多的gag含量。但是到體外培養(yǎng)的后期(21天),我們所檢測的基因表達(dá)和gag含量等都無明顯差異。盡管單純分層軟骨細(xì)胞組相比于相應(yīng)的共培養(yǎng)組沉積更多的gag含量(以dna標(biāo)準(zhǔn)化),但是當(dāng)以初始加入的軟骨細(xì)胞數(shù)量標(biāo)準(zhǔn)化后,第7天時(shí),共培養(yǎng)組相比于相應(yīng)單純軟骨細(xì)胞組沉積相似的gag含量。而第21天時(shí),以初始加入的軟骨細(xì)胞數(shù)量標(biāo)準(zhǔn)化后,共培養(yǎng)組相比于相應(yīng)單純軟骨細(xì)胞組反而沉積更多的gag含量。類似的是,早期(第1、7天)表層軟骨細(xì)胞組和表層軟骨細(xì)胞共培養(yǎng)組分別相比于中深層軟骨細(xì)胞組和中深層軟骨細(xì)胞共培養(yǎng)組分泌更多的lubricin。但是體外培養(yǎng)第21天時(shí),軟骨細(xì)胞組和軟骨細(xì)胞共培養(yǎng)組之間的lubricn濃度無明顯差異。結(jié)論:1.表層和中深層軟骨細(xì)胞分別與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)后能夠在體外三維培養(yǎng)早期(第1、7天)維持不同層軟骨細(xì)胞的分泌細(xì)胞外基質(zhì)的能力;2.但是隨著體外培養(yǎng)時(shí)間的延長(21天),這種分泌細(xì)胞外基質(zhì)能力的差異逐漸消失。第三部分不同層軟骨細(xì)胞與骨髓間充質(zhì)干細(xì)胞三維共培養(yǎng)小珠裸鼠皮下植入后細(xì)胞外基質(zhì)成分的變化目的:1.將海藻酸鈉水凝膠三維共培養(yǎng)小珠植入裸鼠皮下異位成軟骨;2.分別在第2、8周時(shí),處死裸鼠,進(jìn)行組織學(xué)和生物化學(xué)檢測(DNA和GAG定量)。方法:將海藻酸鈉凝膠小珠分別植入到14只4-6周齡裸鼠皮下。分別于第2周和第8周處死裸鼠,取出海藻酸鈉小珠,進(jìn)行DNA和GAG定量以及免疫熒光等檢測。結(jié)果:與體外培養(yǎng)相似,體內(nèi)第2周時(shí),中深層軟骨細(xì)胞共培養(yǎng)組相比于表層軟骨細(xì)胞共培養(yǎng)組沉積更多的GAG。以初始加入的軟骨細(xì)胞數(shù)量標(biāo)準(zhǔn)化后,第2周時(shí)共培養(yǎng)組相比于相應(yīng)單純軟骨細(xì)胞組反而沉積更多的GAG成分。免疫熒光結(jié)果顯示,體內(nèi)第2周時(shí)表層軟骨細(xì)胞組和表層軟骨細(xì)胞共培養(yǎng)組分別相比于中深層軟骨細(xì)胞組和中深層軟骨細(xì)胞共培養(yǎng)組表達(dá)更高水平的Lubricin。但是二型膠原免疫熒光染色的熒光信號(hào)強(qiáng)度卻沒有明顯差異,這也與體外實(shí)驗(yàn)結(jié)果相符合。與體外培養(yǎng)不完全一樣的是,體內(nèi)培養(yǎng)后期(第8周),共培養(yǎng)組和單純軟骨細(xì)胞組都沉積了相似的GAG成分(以DNA標(biāo)準(zhǔn)化)。而以初始加入的軟骨細(xì)胞數(shù)量標(biāo)準(zhǔn)化后發(fā)現(xiàn),共培養(yǎng)組沉積的GAG含量明顯高于單純軟骨細(xì)胞組。此外,體內(nèi)培養(yǎng)第8周時(shí)免疫熒光染色結(jié)果發(fā)現(xiàn)在共培養(yǎng)組之間Lubricin和二型膠原熒光信號(hào)無明顯差異。同時(shí)通過熒光觀察發(fā)現(xiàn),體內(nèi)培養(yǎng)相比于體外培養(yǎng)呈現(xiàn)出更為明顯的細(xì)胞與細(xì)胞之間的直接接觸。結(jié)論:1.表層軟骨細(xì)胞共培養(yǎng)組和中深層軟骨細(xì)胞共培養(yǎng)組在體內(nèi)成軟骨早期(第2周),能夠維持細(xì)胞外基質(zhì)分泌能力的差異;2.在體內(nèi)成軟骨后期(第8周),共培養(yǎng)組之間細(xì)胞外基質(zhì)成分分泌能力的差異消失。
[Abstract]:Background: normal articular cartilage is an uneven stratified tissue, which is essential to maintain the structure and function of articular cartilage. Autologous cartilage transplantation is considered to be the most commonly used and most effective method for the treatment of cartilage defects. However, autologous chondrocyte transplantation does not make the natural difference between the different layers of articular cartilage. More recently, more and more studies have focused on regeneration of stratified cartilage rather than just homogeneous cartilage tissue. It is one of the means to regenerate the corresponding stratification with different layers of chondrocytes. Chondrocytes in different layers can be separated by histological level and can maintain a certain level of delamination in vitro. In vitro, it is found that cartilage tissue using different layers of chondrocytes to regenerate the corresponding layer of cartilage can maintain the corresponding characteristics of different layers. However, it is difficult to regain the stratification characteristic again, and it is difficult to regain the stratification characteristic again. Studies on the repair of cartilage damage by bone cells use different layers of chondrocytes of low generation or non amplification. However, the clinical primary chondrocytes must be amplified by a few generations to obtain sufficient quantities. Therefore, a method can be used to regenerate the layers of different layers of chondrocytes that are short or not expanded. Cartilage, chondrocytes and mesenchymal stem cells co culture and repair cartilage damage, which has been proved to be a promising alternative to repair cartilage damage by simple chondrocytes. The co culture of low generation or unexpanded chondrocytes from the bone marrow mesenchymal stem cells obtained from the non weight-bearing area of the joint has been proved to be capable of producing chondrocytes with individual chondrocytes. A similar effect of cartilage. Therefore, this method can reduce the demand for cartilage cells, which can be used to repair cartilage damage by using short or even non amplification cartilage cells, which still maintain stratification due to the absence of dedifferentiation. In addition, the use of co culture technology may not require cartilage. When cells expand, the repair of cartilage damage can even become a step. However, it is still unclear whether the two layers of chondrocytes can maintain the characteristics of different layers together after co culture with bone marrow mesenchymal stem cells. In this study, we use a commonly used in vitro three-dimensional culture system, sodium alginate hydrogel. The external culture system is used to study the characteristics of different layers of chondrocytes and bone marrow mesenchymal stem cells in vitro and in vitro. The first part is the separation of different layers of cartilage cells and the maintenance of stratification characteristics in the monolayer culture environment: 1. extraction of surface and middle layer cartilage cells; 2. The gene expression between the representative layer and the medium deep cartilage cells, the difference in the protein synthesis and proliferation rate; 3. the gene expression between the surface and the middle deep cartilage cells after the passage was detected, the difference in the protein synthesis and proliferation rate; and 4. to separate and verify the bone marrow mesenchymal stem cells. Method: the 1 month old New Zealand rabbit knee was extracted with a microscoper. The cartilaginous tissue about 100um thick on the surface of the femur surface is the source of the surface cartilage cells. The remaining cartilage tissue is the source of the medium deep cartilage cells. The original surface and middle deep cartilage cells are obtained through the digestion of type two collagenase. The bone marrow of the femur and tibia in 1 month old New Zealand rabbits is extracted and the properties of the bone marrow mesenchymal stem cells are applied to the wall. Bone marrow mesenchymal stem cells were obtained. The adherent bone marrow mesenchymal stem cells showed multiple differentiation ability through multi direction induction. Col2a, acan and PRG4 genes were detected for the original and middle deep cartilage cells (P0 generation). GAPDH was used as the internal parameter and 2- delta delta CT method was used to compare them. The original representative layer and the middle and deep cartilage were compared. Cells were added to the 96 orifice plate for monolayer cell proliferation experiment. On day 3,5,7,10, CCK-8 (cellcountingkit-8) was used to detect the proliferation rate. The original representative layer and the medium deep cartilage cells were defined as the P1 generation when the fusion was long to 90%. By this analogy, the same gene and protein levels were detected for the P1 and P2 generations, and the results of the proliferation rate were detected. It was found that there were obvious differences between the primary and middle layer cartilage cells, that is, the higher expression of PRG4 gene and col2a gene in the surface chondrocytes, and the higher expression of acan gene in the medium deep cartilage cells. The difference in gene expression and protein level gradually diminished and the difference in P2 generation disappeared. The extracted bone marrow cells were fibroblast like cells, and the third generations of bone marrow mesenchymal stem cells could be induced to adipocytes, osteoblasts and chondrocytes. Conclusion: 1. by histologically separated P0 representative layer soft. Bone cells and medium deep cartilage cells showed different characteristics; 2. with the generation, the gene expression of different layers of chondrocytes, protein level and proliferation velocity difference gradually disappeared. Second the changes of extracellular matrix components of different layers of chondrocytes and bone marrow mesenchymal stem cells in vitro: 1. use the sea in vitro The three dimensional culture system of sodium alginate hydrogel co cultured the surface and middle depth chondrocytes with bone marrow mesenchymal stem cells respectively. 2. the expression of gene expression in the extracellular matrix of cartilage was detected at different time points (day 1,7,21). 3. the content of the main component of cartilage extracellular matrix (GAG) at different time points (day 1,7,21) and the content of DNA were changed. Method: after mixing the P0 representative or middle - deep cartilage cells alone or with bone marrow mesenchymal stem cells in the 1:2 ratio, the concentration of 6 x 106/ml cells was mixed with 2% sodium alginate, and the sodium alginate hydrogel was dripped by a drop of syringe into the coagulated droplet in 102mm calcium chloride (102mm calcium chloride). In the culture medium of cartilage induced. The gene expression and gag, DNA content were detected at day 1,7,21. The lubricin concentration was measured by ELISA method for 24 hours culture of culture day 1,7,21. Results: in the first day and seventh day of gene expression, the medium depth chondrocyte co culture group was compared with the surface cartilage. The co culture group expressed a higher acan gene and a lower PRG4 gene, but there was no significant difference in the level of col2a. The same trend was also found between the different layers of chondrocytes. The medium depth chondrocyte co culture group and the medium depth chondrocyte group deposited more GA than the surface chondrocyte co culture group and the surface chondrocyte group at seventh days. G content. But there was no significant difference in gene expression and GAG content in the later period of culture (21 days). Although the simple stratified chondrocyte group had more GAG content than the corresponding co culture group (DNA Standardization), the co culture group was at seventh days when the number of initial chondrocytes was standardized. Compared to the corresponding simple chondrocyte group, the content of gag was similar to that of the corresponding chondrocyte group. On the twenty-first day, the co culture group deposited more GAG content than the corresponding chondrocyte group when the number of initial chondrocytes was standardized. Similarly, the early (day 1,7) surface chondrocyte and surface chondrocyte co culture group compared with the coculture group. More lubricin. was secreted in the medium depth chondrocyte group and the medium depth chondrocyte co culture group, but there was no significant difference in the concentration of lubricn between the chondrocyte and chondrocyte co culture groups for twenty-first days in vitro culture. Conclusion: the 1. surface and middle deep cartilage cells co culture with bone marrow mesenchymal stem cells can be in vitro three after the co culture of bone marrow mesenchymal stem cells. The ability to secrete the extracellular matrix of different layers of chondrocytes at early stage (day 1,7); 2. but with the extension of the time of culture in vitro (21 days), the difference between the extracellular matrix capacity of the secretory cells gradually disappeared. Third parts of different layers of chondrocytes and bone marrow mesenchymal stem cells were co cultured with subcutaneous implanted cells after subcutaneous implantation of nude mice. Changes in the components of the external matrix: 1. the three dimensional co cultured beads of sodium alginate hydrogel were implanted subcutaneously into the subcutaneous subcutaneous cartilage in nude mice; 2. the nude mice were killed at week 2,8, and the tissue and biochemical tests (DNA and GAG quantitative) were performed. Methods: the sodium alginate gel beads were subdivided into 14 4-6 weeks old nude mice. Eighth weeks of death, the nude mice were killed, sodium alginate beads were removed, DNA and GAG quantitative and immunofluorescence were detected. Results: similar to in vitro culture, at second weeks in the body, the medium and deep cartilage cell co culture group deposited more GAG. than the surface chondrocyte co culture group, and after the initial addition of chondrocytes, the co culture was co cultured at second weeks. More GAG components were deposited in the group compared with the corresponding simple chondrocyte group. The immunofluorescence results showed that the surface chondrocyte group and the surface chondrocyte co culture group at second weeks showed a higher level of Lubricin. but two collagen immunofluorescence compared with the medium deep cartilage cell group and the medium deep cartilage cell co culture group. The fluorescence intensity of the light staining was not significantly different, which was also in accordance with the experimental results in vitro. The similar GAG component (DNA Standardization) was deposited in the co culture group and the simple chondrocyte group in the late culture (eighth weeks). The content of GAG in the culture group was significantly higher than that in the simple chondrocyte group. In addition, the immunofluorescence staining results showed that there was no significant difference between Lubricin and type two collagen fluorescent signals between the co culture groups at eighth weeks. Conclusion: 1. the co culture group and the medium deep cartilage cell co culture group and the middle depth chondrocyte co culture group can maintain the difference in the secretion of extracellular matrix in the early stage (second weeks), and 2. in the late stage of the cartilage (eighth weeks), the difference of the secretory ability of the extracellular matrix between the co culture groups disappeared.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R68

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