【摘要】：目的： 一、利用京尼平交聯(lián)脫氧膽酸鈉酶聯(lián)合法與Trixon-100酶聯(lián)合法,分別結(jié)合BMMSCs制備組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物。 二、對(duì)組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物的急性毒性、細(xì)胞毒性、血管化能力、體內(nèi)免疫炎性反應(yīng)及支架降解被吸收情況檢測(cè)評(píng)價(jià)。 三、通過(guò)對(duì)組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物的生物安全性能進(jìn)行評(píng)價(jià),為制備更接近原生氣管的替代物提供實(shí)驗(yàn)依據(jù)。 方法： 一、組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物的制備： ①細(xì)胞部分：將2月齡大體重約1.2kg的健康新西蘭大白兔一只作為BMMSCs供體,經(jīng)原代及傳代培養(yǎng)獲得第3代BMMSCs。 ②脫細(xì)胞氣管支架的制備：將5只4月齡體重在2.5kg健康新西蘭大白兔作為氣管支架的供體,分別用京尼平交聯(lián)脫氧膽酸鈉酶聯(lián)合法與Trixon-100酶聯(lián)合法對(duì)取材的氣管進(jìn)行脫細(xì)胞處理獲得脫細(xì)胞氣管支架。 ③脫細(xì)胞氣管支架細(xì)胞復(fù)合物的制備：將BMMSCs分別種植于上述兩種氣管支架外表面并培養(yǎng)。 ④實(shí)驗(yàn)分組：京尼平交聯(lián)脫氧膽酸鈉酶聯(lián)合法結(jié)合BMMSCs (5x104cells/ml)制備的組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物作為實(shí)驗(yàn)A組,Trixon-100酶聯(lián)合法結(jié)合BMMSCs制備組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物后者作為實(shí)驗(yàn)B組。 二、通過(guò)以下5種方法檢測(cè)組織工程脫細(xì)胞氣管支架細(xì)胞復(fù)合物的生物安全性： ①將A組浸漬液、B組浸漬液及陰性組(生理鹽水)注入小鼠腹腔檢測(cè)兩種脫細(xì)胞氣管支架細(xì)胞復(fù)合物對(duì)小鼠毒性； ②應(yīng)用MTT法檢測(cè)A組、B組、陰性對(duì)照組(聚全氟乙丙烯)及陽(yáng)性對(duì)照組(聚氟乙烯)中BMMSCs生長(zhǎng)增殖情況； ③通過(guò)SEM(掃描電子顯微鏡檢查)來(lái)觀察兩實(shí)驗(yàn)組表面的BMMSCs粘附生長(zhǎng)狀態(tài); ④對(duì)A組、B組、陰性組(戊二醛浸潤(rùn)的A組)及陽(yáng)性組(明膠海綿)應(yīng)用雞胚絨毛膜實(shí)驗(yàn)觀察脫細(xì)胞氣管支架細(xì)胞復(fù)合物的血管化程度； ⑤以原BMMSCs供體大白兔作為移植受體,將這兩實(shí)驗(yàn)組分別于移植于受體皮下脊柱兩側(cè)。移植后第7、14、28d取出脫細(xì)胞氣管支架細(xì)胞復(fù)合物,與移植前的脫細(xì)胞氣管支架細(xì)胞復(fù)合物,分別進(jìn)行連續(xù)形態(tài)學(xué)觀察及HE染色,以檢測(cè)免疫炎癥反應(yīng)。 結(jié)果： 一、脫細(xì)胞氣管支架細(xì)胞復(fù)合物浸漬液注入小鼠腹腔后,三組小鼠活動(dòng)度、進(jìn)食及排便均正常,體重增加(p0.05)及呼吸頻率變化(p0.05)無(wú)統(tǒng)計(jì)學(xué)意義。 二、細(xì)胞形態(tài)學(xué)觀察發(fā)現(xiàn)兩實(shí)驗(yàn)組上的BMMSCs均貼壁生長(zhǎng)良好,無(wú)明顯差異。 三、MTT檢測(cè)繪制細(xì)胞生長(zhǎng)曲線,發(fā)現(xiàn)陰性對(duì)照組細(xì)胞生長(zhǎng)最好,陰性組最差,B組細(xì)胞增長(zhǎng)比A組稍快,但無(wú)明顯區(qū)別。 四、掃描電子顯微鏡：兩組BMMSCs均完全粘附覆蓋支架外表面,細(xì)胞層疊,連接緊密。 五、雞胚絨毛膜實(shí)驗(yàn)觀察顯示：A組、B組及陽(yáng)性組周?chē)?jiàn)新生血管形成,以陽(yáng)性組最明顯,A組新生血管較多,部分新生血管爬行于脫細(xì)胞氣管支架復(fù)合物底面,B組新生血管少于A組,陰性組周?chē)匆?jiàn)明顯血管形成。 六、通過(guò)四次(移植前、移植后第7、14、28d脫細(xì)胞氣管支架細(xì)胞復(fù)合物)連續(xù)組織學(xué)檢測(cè)及HE染色發(fā)現(xiàn)：A組細(xì)胞支架復(fù)合物仍為暗紅色,硬度無(wú)減弱,無(wú)明顯炎癥,細(xì)胞支架復(fù)合物各層結(jié)構(gòu)完整,無(wú)降解；B組細(xì)胞支架復(fù)合物呈淡紅色,硬度減弱,炎癥較重,支架各層結(jié)構(gòu)混亂,逐漸軟化降解。 結(jié)論： 一、A組與B組均無(wú)急性毒性及細(xì)胞毒性反應(yīng),均可促進(jìn)細(xì)胞生長(zhǎng)增殖及支架基質(zhì)血管再生,且A組血管化能力更強(qiáng)。 二、A組不引起炎癥反應(yīng)、不易軟化降解的性能更有利于移植體在體內(nèi)長(zhǎng)期存活及功能作用。
[Abstract]:Objective:
Firstly, the acellular tracheal scaffold cell complex was prepared by Genipin cross-linked deoxycholate sodium enzyme combined with Trixon-100 enzyme combined with BMMSCs.
Secondly, the acute toxicity, cytotoxicity, vascularization ability, in vivo immune inflammatory reaction and scaffold degradation and absorption of acellular tracheal scaffold complex were detected and evaluated.
Thirdly, the biosafety of acellular tracheal scaffold cell complex was evaluated to provide experimental basis for the preparation of substitutes closer to the original gas tube.
Method:
(1) preparation of tissue engineered acellular tracheal scaffold complex:
(1) Cell part: A 2-month-old healthy New Zealand white rabbit weighing about 1.2 kg was used as a BMMSCs donor. The third generation of BMMSCs were obtained by primary culture and subculture.
(2) Preparation of acellular tracheal stents: Five 4-month-old healthy New Zealand white rabbits weighing 2.5 kg were used as tracheal stent donors. The acellular tracheal stents were obtained by acellular treatment with Genipin crosslinked deoxycholate enzyme and Trixon-100 enzyme respectively.
(3) Preparation of acellular tracheal scaffold cell complex: BMMSCs were implanted on the surface of the two kinds of tracheal scaffolds and cultured.
(4) Experimental grouping: The tissue engineered acellular tracheal scaffold cell complex prepared by Genipin cross-linked deoxycholate enzyme combined with BMMSCs (5x104 cells/ml) was used as experimental group A, and the tissue engineered acellular tracheal scaffold cell complex prepared by Trixon-100 enzyme combined with BMMSCs as experimental group B.
Secondly, the biosafety of tissue engineered acellular tracheal scaffold cell complex was detected by the following five methods:
The toxicity of two acellular tracheal scaffold cell complexes was detected by injecting group A, group B and negative group (normal saline) into the abdominal cavity of mice.
(2) The growth and proliferation of BMMSCs in group A, group B, negative control group (PFE) and positive control group (PFE) were detected by MTT.
(3) observe the adherence growth state of BMMSCs on the two experimental group by SEM (scanning electron microscopy).
(4) The vascularization of acellular tracheal scaffold cell complex was observed in group A, group B, negative group (glutaraldehyde infiltration group A) and positive group (gelatin sponge).
_The two experimental groups were transplanted into the subcutaneous spine of the recipient with the original BMMSCs donor rabbit as the recipient. On the 7th, 14th and 28th day after transplantation, the acellular tracheal scaffold cell complex was taken out and the acellular tracheal scaffold cell complex was observed and stained with HE to detect the immunoinflammatory reaction.
Result:
Firstly, after the acellular tracheal scaffold cell complex impregnated solution was injected into the abdominal cavity of mice, the activity, food intake and defecation of the three groups of mice were normal, weight gain (p0.05) and respiratory rate changes (p0.05) were not statistically significant.
Two, cell morphology observation showed that two of BMMSCs in experimental group adhered well and had no significant difference.
Third, MTT assay plotted the cell growth curve, found that the negative control group had the best cell growth, the negative group had the worst cell growth, the B group cell growth was slightly faster than the A group, but there was no significant difference.
Fourthly, scanning electron microscopy: BMMSCs in both groups completely adhered to the outer surface of the scaffold, and the cells overlapped tightly.
Fifth, the chicken embryo chorionic membrane experiment showed that neovascularization was observed in group A, group B and around the positive group. The most obvious neovascularization was found in the positive group. There were more neovascularization in group A. Some of the neovascularization crawled on the bottom of the acellular tracheal stent complex.
Sixthly, four times (before transplantation, 7, 14, 28 days after transplantation) continuous histological examination and HE staining showed that the scaffold complex in group A was still dark red, the stiffness was not weakened, there was no obvious inflammation, the structure of the scaffold complex was intact, and there was no degradation; the scaffold complex in group B was light red, and the stiffness was reduced. The structure of the scaffold is chaotic and gradually softened and degraded.
Conclusion:
First, both group A and group B had no acute toxicity and cytotoxicity. Both groups could promote cell growth and proliferation and angiogenesis of scaffold matrix, and group A had stronger vascularization ability.
Secondly, group A did not cause inflammation and was not easy to soften and degrade, which was more conducive to the long-term survival and function of the graft in vivo.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R318.08

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