【摘要】：脊髓損傷是臨床常見的高致殘率的疾病,由于損傷神經(jīng)元的再生能力極其微弱,加之微環(huán)境中抑制性因素的存在,導致脊髓損傷后的神經(jīng)再生及功能恢復(fù)非常有限。目前脊髓損傷的治療理念是重塑損傷脊髓的空間結(jié)構(gòu),改善神經(jīng)元再生環(huán)境進而誘導軸突再生,組織工程是實現(xiàn)這一理念的有效途徑。本研究應(yīng)用脫細胞神經(jīng)支架復(fù)合胎盤間充質(zhì)干細胞(PMSCs),構(gòu)建組織工程化神經(jīng)支架復(fù)合體,治療大鼠脊髓橫斷性缺損,為臨床應(yīng)用組織工程治療脊髓損傷提供理論基礎(chǔ)。首先,選擇改良脫細胞方法,制備理想的脫細胞神經(jīng)支架。行HE染色、甲苯胺藍染色、Laminin免疫熒光染色,觀察脫細胞神經(jīng)支架組織形態(tài)特征；掃描電鏡、透射電鏡觀察脫細胞神經(jīng)支架超微結(jié)構(gòu),DNA含量測定檢測脫細胞神經(jīng)支架細胞殘留,細胞增殖率和肌袋埋入反應(yīng)檢測支架的細胞毒性和生物相容性。隨后,構(gòu)建組織工程化復(fù)合體,應(yīng)用散點注射法將PMSCs注入脫細胞神經(jīng)支架,構(gòu)建細胞支架復(fù)合體,37℃,5%C02培養(yǎng)箱中培養(yǎng)。最后,制作大鼠橫斷性脊髓缺損模型,移植神經(jīng)支架復(fù)合體,術(shù)后4W,免疫熒光檢鋇PMSCs在體生長、遷移及分化情況,BBB評分、腳印分析及電生理檢測損傷大鼠的功能恢復(fù),透射電鏡觀察軸突及髓鞘再生。HE染色、甲苯胺藍染色、透射電鏡結(jié)果顯示,改良法制備的脫細胞神經(jīng)支架,細胞脫除徹底,ECM蛋白含量豐富；掃描電鏡結(jié)果顯示,支架三維空間結(jié)構(gòu)完整,管道通暢；細胞毒性檢測結(jié)果顯示,支架無胞毒性；生物組織相容性檢測結(jié)果顯示,無炎癥細胞浸潤,細胞相容性檢測結(jié)果顯示,細胞長入支架并大量存活,生長狀態(tài)良好。神經(jīng)支架復(fù)合體移植術(shù)后4W,取材行冰凍切片,熒光顯微鏡觀察顯示,PMSCs生長狀態(tài)良好,向損傷區(qū)兩端雙向遷移,兔疫熒光結(jié)果顯示植入的PMSCs分化為神經(jīng)元細胞。透射電鏡觀察顯示,與對照組相比,支架復(fù)合體組大鼠新生髓鞘數(shù)目明顯增多,厚度明顯增厚,直徑明顯增大；BBB評分顯示,支架復(fù)合體組大鼠BBB評分明顯提高,術(shù)后8w達到13分；腳印分析測定表明支架復(fù)合體組大鼠前后足協(xié)調(diào)性明顯改善,術(shù)后8w, ILC、AR分別為1.8,12；電生理檢測結(jié)果顯示,支架復(fù)合體組運動誘發(fā)電位振幅為0.54±0.05mV,潛伏期為9.98±0.33ms, PMSCs組可記錄到輕微電信號,生理鹽水組無電信號記錄。綜上所述,改良法制備的脫細胞神經(jīng)支架,是構(gòu)建神經(jīng)支架復(fù)合體的理想材料；改良脫細胞支架復(fù)合PMSCs構(gòu)建神經(jīng)支架復(fù)合體,治療大鼠橫斷性脊髓損傷,有助于促進大鼠功能恢復(fù)；本研究為臨床上應(yīng)用組織工程化橋接神經(jīng)元治療脊髓損傷提供理論基礎(chǔ)和技術(shù)支撐。
[Abstract]:Spinal cord injury (sci) is a common disease with high disability rate. Because of the very weak regeneration ability of injured neurons and the existence of inhibitory factors in microenvironment, the regeneration and functional recovery of injured neurons are very limited. At present, the idea of spinal cord injury treatment is to reconstruct the spatial structure of injured spinal cord, improve the regeneration environment of neurons and induce axon regeneration. Tissue engineering is an effective way to realize this idea. In this study, acellular nerve scaffolds combined with placental mesenchymal stem cells (PMSCs),) were used to construct tissue-engineered nerve scaffolds for the treatment of spinal cord transverse defects in rats, which provided a theoretical basis for clinical application of tissue engineering in the treatment of spinal cord injury. Firstly, an improved acellular method was chosen to prepare an ideal acellular nerve scaffold. He staining, toluidine blue staining and laminin immunofluorescence staining were used to observe the morphological characteristics of acellular nerve scaffolds, and the ultrastructure of acellular nerve stents was observed by scanning electron microscope and transmission electron microscope. The cytotoxicity and biocompatibility of the scaffold were examined by cell proliferation rate and muscle bag embedding reaction. Then, the tissue engineering complex was constructed, PMSCs was injected into acellular nerve scaffold by scattered point injection, and the cell scaffold complex was cultured in 52 incubator at 37 鈩，

本文編號：2177714