同種異體骨髓間充質(zhì)干細(xì)胞復(fù)合膠原海綿修復(fù)兔橈骨缺損的實(shí)驗(yàn)研究

1、同種異體骨髓間充質(zhì)干細(xì)胞復(fù)合膠原海綿修復(fù)兔橈骨缺損的實(shí)驗(yàn)研究         11-02-13 13:33:00     編輯：studa20              作者：楊柏林，潘勇，云雄，鄒重文，曹國永【摘要】  目的：了解兔骨髓間充質(zhì)干細(xì)胞(BMscs)同種異體移植的存活分布情況及同種異體兔BMscs復(fù)合膠原海綿(CS)情況，觀

2、察其對(duì)兔橈骨1.5 cm缺損的修復(fù)效果，為將來的進(jìn)一步應(yīng)用奠定實(shí)驗(yàn)基礎(chǔ)。方法：采集、培養(yǎng)、純化擴(kuò)增新西蘭大白兔BMscs，用eGFP熒光標(biāo)記BMscs，與膠原海綿聯(lián)合培養(yǎng)，植于同種異體動(dòng)物臀大肌，觀察存活及分布情況。建立兔橈骨缺損(1.5 cm)模型，45只新西蘭大白兔隨機(jī)分為3組：同種異體BMscs/CS組(A組)，單純膠原海綿組(B組 )，空白對(duì)照組(C組)。術(shù)后分階段行X線、組織學(xué)檢查及生物力學(xué)檢查，評(píng)價(jià)其修復(fù)效果。結(jié)果：eGFP標(biāo)記的BMscs異體移植存活時(shí)間超過3周。X線示12周A組大白兔5只中有4只達(dá)到骨性連接。新生骨量呈A組C組遞減，至12周無一愈合。隨著時(shí)間推移，A組編織骨髓腔

3、和骨小梁增多，修復(fù)過程優(yōu)于B、C組。A組生物力學(xué)指標(biāo)均低于健側(cè)正常對(duì)照組，但各指標(biāo)能達(dá)到正常組的80%90%。結(jié)論：同種異體MSCs復(fù)合CS可以修復(fù)1.5 cm兔橈骨缺損。 【關(guān)鍵詞】  骨髓間充質(zhì)干細(xì)胞;組織工程;移植;骨缺損             ABSTRACT Objective:To study the survival and distribution of allogenic mesenchymal stem cells (BMscs) a

4、fter implantation into other rabbits, evaluate osteogenetic effectiveness of collagensponge mixed with allogenic BMscs of rabbit in repairing of radial shaft defects, and lay the foundation for further application.Methods: The BMscs of New Zealand white rabbits were collected，cultured，purified and a

5、mplified，they were marked with eGFP, cultured with collagensponge and planted into ectogluteus of other rabbits in order to observe the survival and distribution of BMscs. Model of radial bone defects (15 mm )of rabbits was established. 45 rabbits were divided into 3 groups, group A (BMscs/CS group)

6、, group B (CS group) and group C (untreated group). Roentgenographic, histologic, biomechanics indexed of the animals were examined at certain time after surgery to evaluate osteogenetic effectiveness.Results: The BMscs marked with eGFP could survive for 3 weeks. On the 12th week, 4 of 5 radial bone

7、 defects healed in group A.The quantity of callus decreased from group A to C. In group B and C, no one rabbits healed on the 12th week. Biomechanics result showed the indexes of group A were lower than the opposite side normal control groups.Conclusion: BMscs mixed with CS can repair radial defect

8、shorter than 1.5 cm in rabbit.KEY WORDS Bone marrow mesenchymal stem cells; Tissue engineering; Transplant; Bone defects利用組織工程的方法治療骨缺損，是目前骨科基礎(chǔ)研究的熱點(diǎn)之一。目的基因、種子細(xì)胞、支架材料是組織工程三大要素，種子細(xì)胞作為其中一項(xiàng)要素引起了廣泛的研究1，2，其中骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells， BMscs )是研究較多的種子細(xì)胞之一。傳統(tǒng)的自體細(xì)胞移植在實(shí)驗(yàn)室研究階段存在著大量細(xì)胞取材困難，宿主存活時(shí)間

9、難以保證，不同宿主細(xì)胞容易混雜污染等缺點(diǎn)。研究發(fā)現(xiàn)BMscs可有效繞過機(jī)體的免疫排斥進(jìn)行異體移植，基于此本文將異體BMscs復(fù)合膠原海綿，對(duì)兔1.5 cm橈骨缺損修復(fù)進(jìn)行了實(shí)驗(yàn)研究。1 材料與方法1.1 材料優(yōu)質(zhì)胎牛血清(杭州四季青公司);DMSO(上海生化試劑二廠);MTT(美國Sigma公司);Percoll分離液(密度1.073 g/mL，天津TDB公司);DMEM/F12培養(yǎng)基(美國Gibco公司);胰蛋白酶(美國Amesco公司);注射用肝素鈉、注射用鏈霉素、速眠新(華北制藥股份有限公司);膠原海綿(北京益而康公司)新西蘭大白兔(第三軍醫(yī)大學(xué)大坪醫(yī)院動(dòng)物中心);eGFPAd由重慶武警

10、總隊(duì)醫(yī)院曹國永博士提供。1.2 方法將上述方法獲得的eGFPAdBMscsCS復(fù)合材料取出，PBS清洗，2.5%戊二醛固定，乙醇和丙酮梯度脫水，和CS一起臨界點(diǎn)干燥后噴金，掃描電鏡觀察。評(píng)分標(biāo)準(zhǔn)如下：新生骨面積百分比1%24%計(jì)1分;25%49%計(jì)2分;50%74%計(jì)3分;75%99%計(jì)4分;100%計(jì)5分。2 結(jié)果2.1 原代BMscs 顯微鏡觀察結(jié)果BMSC形態(tài)與成纖維細(xì)胞類似，呈紡錘形，有少量細(xì)胞突起。隨著換液的進(jìn)行，出現(xiàn)集落狀細(xì)胞群，由1020個(gè)細(xì)胞組成，此后細(xì)胞集落迅速增多，融合成片，呈旋渦狀排列， 912 d細(xì)胞鋪滿瓶底成單層，見圖1。圖1 倒置顯微鏡下原代BMscs圖象(×100)2.2 膠原海綿及eGFPAdBMscsCS復(fù)合體掃描電鏡觀察結(jié)果膠原海綿疏松多孔，大小不均。復(fù)合材料掃描觀察見BMscs在CS支架材料上伸展良好，細(xì)胞融合成片狀，細(xì)胞與周圍材料緊密相連，少數(shù)成球形(圖2)。2.3 eGFP標(biāo)記的BMscs體內(nèi)示蹤觀察結(jié)果術(shù)后3 d，冰凍切片在熒光顯微鏡下可見移植區(qū)大量eGFP陽性細(xì)胞。術(shù)后7 d可見少許減弱，在膠原海綿外周邊組織亦發(fā)現(xiàn)有少許eGFP陽性細(xì)胞。