發(fā)布時(shí)間：2018-01-07 18:43

  本文關(guān)鍵詞：脊髓損傷的臍帶間充質(zhì)干細(xì)胞治療的實(shí)驗(yàn)研究及臨床不同治療方案的療效對(duì)比研究　出處：《河北醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 頸脊髓損傷 臍帶間充質(zhì)干細(xì)胞 小鼠 無骨折脫位 前路減壓 后路減壓 療效比較

【摘要】：第一部分臍帶間充質(zhì)干細(xì)胞分化為神經(jīng)細(xì)胞并修復(fù)脊髓損傷的研究目的:脊髓損傷(spinal cord injury,SCI)是由外傷誘發(fā)的一種脊柱外科創(chuàng)傷性疾病,表現(xiàn)為在損傷節(jié)段以下出現(xiàn)感覺、運(yùn)動(dòng)和自主神經(jīng)功能障礙。國(guó)外流行病學(xué)調(diào)查顯示,每年全球有13萬新發(fā)脊髓損傷患者,且有超過250萬患者正飽受著不同程度的脊髓損傷后遺癥困擾,而這些SCI患者每年的醫(yī)療支出將超過60億美元,給家庭及社會(huì)造成沉重負(fù)擔(dān)。目前臨床上用于治療SCI的方法主要局限在藥物、手術(shù)及各種物理干預(yù),SCI的干細(xì)胞治療仍停留在動(dòng)物相關(guān)的基礎(chǔ)研究層面,如骨髓基質(zhì)干細(xì)胞移植、嗅鞘細(xì)胞移植、臍帶血間充質(zhì)干細(xì)胞移植、臍帶華通膠來源間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)移植、Nogo A疫苗移植等。由于臍帶間充質(zhì)干細(xì)胞具有來源廣泛、易擴(kuò)增、無倫理學(xué)方面的限制,已成為基礎(chǔ)實(shí)驗(yàn)中用于治療脊髓損傷的新熱點(diǎn)和新方向。臍帶血間充質(zhì)干細(xì)胞在特定的環(huán)境誘導(dǎo)下可以分化為具有不同功能的組織細(xì)胞,并促進(jìn)保護(hù)性因子的產(chǎn)生,且具有整合、調(diào)節(jié)、遷移和分泌等作用。當(dāng)分化為神經(jīng)細(xì)胞后可以促進(jìn)神經(jīng)組織的修復(fù),改善脊髓損傷大鼠的神經(jīng)功能。國(guó)內(nèi)有學(xué)者采用移植人臍帶血干細(xì)胞的方法來治療大鼠脊髓損傷,雖獲得了滿意的療效,但存在干細(xì)胞分化效率較低等問題。本研究旨在提高臍帶間充質(zhì)干細(xì)胞體外誘導(dǎo)分化為神經(jīng)細(xì)胞的效率,并將其移植到脊髓損傷小鼠動(dòng)物模型,為脊髓損傷這一難治性疾病提供了理論基礎(chǔ)。方法:采集人臍帶并采用植塊法、膠原酶消化法及膠原酶與胰酶聯(lián)合消化法分離人臍帶間充質(zhì)干細(xì)胞(Human umbilical cord mesenchymal stem cells,h UCMSCs),培養(yǎng)、擴(kuò)增后,將Nurr-1轉(zhuǎn)染到臍帶間充質(zhì)干細(xì)胞,并聯(lián)合雞尾酒因子(ATRA,GGF-2,b FGF,PDGF and forskolin)將臍帶間充質(zhì)干細(xì)胞誘導(dǎo)分化為神經(jīng)細(xì)胞,利用免疫熒光技術(shù)檢測(cè)神經(jīng)細(xì)胞標(biāo)志物表達(dá)情況。取昆明系小白鼠進(jìn)行脊髓損傷模型的建立,并將分化后的細(xì)胞移植到脊髓損傷小鼠體內(nèi),免疫組化檢測(cè)細(xì)胞移植后的定植情況,判斷其治療效果。結(jié)果:人臍帶間充質(zhì)干細(xì)胞經(jīng)過誘導(dǎo)后,細(xì)胞開始收縮變圓,進(jìn)而變得拉伸后成為多角樣,在持續(xù)增長(zhǎng)的過程中,細(xì)胞的胞體開始拉絲樣出現(xiàn)神經(jīng)細(xì)胞的突觸樣結(jié)構(gòu)。誘導(dǎo)2周后,借助免疫熒光檢測(cè)法可以檢測(cè)出神經(jīng)細(xì)胞特有標(biāo)志物MAP-2和MBP存在,且經(jīng)Nurr-1過表達(dá)的臍帶間充質(zhì)干細(xì)胞分化為神經(jīng)細(xì)胞的效率最高。造模小鼠的脊髓損傷段結(jié)構(gòu)遭到破壞,灰質(zhì)內(nèi)出現(xiàn)較多的神經(jīng)元死亡,前角部分神經(jīng)元尚存,其中部分神經(jīng)元可見核固縮。h UCMSCs移植后細(xì)胞在損傷部位大量增殖,熒光顯微鏡下可檢測(cè)到抗人細(xì)胞核陽(yáng)性細(xì)胞,術(shù)后小鼠BBB評(píng)分逐漸提高,后肢運(yùn)動(dòng)功能逐漸改善。結(jié)論:經(jīng)Nurr-1基因轉(zhuǎn)染的臍帶間充質(zhì)干細(xì)胞在雞尾酒因子的誘導(dǎo)下可以獲得最好的分化及增殖,移植到受傷脊髓處可以在損傷部位增殖,同時(shí)小鼠的后肢功能獲得明顯改善,表明治療脊髓損傷療效確切。第二部分無骨折脫位型脊髓損傷手術(shù)與非手術(shù)治療的臨床療效對(duì)比研究目的:無骨折脫位型脊髓損傷(cervical spinal cord injury without fracture or dislocation,CSCIWFD)是一類特殊類型的脊髓損傷,臨床較多間,通常指頸部受到“較輕微”外力損傷后經(jīng)過影像學(xué)檢查,如X線、CT掃描等均未發(fā)現(xiàn)明顯的頸椎骨折或脫位,但脊髓損傷癥狀相對(duì)較重的一類骨科疾病,大約占到頸脊髓損傷總數(shù)的23%。查閱文獻(xiàn)發(fā)現(xiàn),CSCIWFD的治療方法主要分為兩種,一種是采用大劑量糖皮質(zhì)激素沖擊、神經(jīng)營(yíng)養(yǎng)藥物靜點(diǎn)及高壓氧治療的保守治療(非手術(shù)治療),另一類是積極采用外科手段對(duì)脊髓進(jìn)行充分減壓的手術(shù)治療。每種治療方法都可以促使CSCIWFD患者獲得一定程度的神經(jīng)功能恢復(fù),但手術(shù)干預(yù)作為一種有創(chuàng)性操作,其總體療效是否優(yōu)于非手術(shù)治療呢?針對(duì)此問題,我們進(jìn)行了較深入的臨床對(duì)比研究。方法:回顧性分析2013年10月~2015年9月在門診就診和住院治療的110例CSCIWFD患者的臨床資料,其中92例獲得了超過18個(gè)月的臨床隨訪,根據(jù)患者所接受的治療方法分為兩組:A組(非手術(shù)組),38例,其中男23例,女15例,年齡19~63歲,平均42.1歲,均接受非手術(shù)治療;B組(手術(shù)組),54例,其中男35例,女19例,年齡17~62歲,平均40.8歲,均接受手術(shù)治療。記錄手術(shù)治療組患者的手術(shù)時(shí)間、術(shù)中出血量及并發(fā)癥發(fā)生情況;兩組分別在治療前、治療后1周、治療后1個(gè)月及末次隨訪時(shí)采用日本骨科協(xié)會(huì)(Japanese Orthopaedic Association,JOA)脊髓損害評(píng)分表(17分法)評(píng)估治療效果;末次隨訪時(shí)根據(jù)ASIA脊髓損傷分級(jí)標(biāo)準(zhǔn)對(duì)兩組患者進(jìn)行比較分析。結(jié)果:B組所有患者均順利接受手術(shù),其中10例患者接受頸前路椎間盤切除植骨內(nèi)固定術(shù),17例患者接受頸前路椎體次全切除內(nèi)固定術(shù),16例患者接受頸后路單開門椎管擴(kuò)大成形鈦板固定術(shù),11例患者接受頸后路全椎板減壓側(cè)塊螺釘固定術(shù)。平均手術(shù)時(shí)間175.2 min,平均出血量為210.6 ml。A組JOA評(píng)分由治療前的7.0±1.8分提高至術(shù)后1周時(shí)的7.9±2.0分、1個(gè)月時(shí)的9.1±2.2分、3個(gè)月時(shí)的9.9±2.3分、6個(gè)月時(shí)的10.5±2.4分和末次隨訪時(shí)的11.2±2.9分,治療前后比較差異具有顯著性(P0.05),神經(jīng)功能改善率為41.3%±9.6%;B組JOA評(píng)分由術(shù)前的6.7±1.6分提高至術(shù)后1周時(shí)的8.9±2.2分、1個(gè)月時(shí)的10.6±2.5分、3個(gè)月時(shí)的12.0±2.8分、6個(gè)月時(shí)的13.9±3.1分和末次隨訪時(shí)的14.5±3.6分,手術(shù)前后比較具有顯著統(tǒng)計(jì)學(xué)意義(P0.05),神經(jīng)功能改善率為70.2%±10.3%。末次隨訪時(shí)根據(jù)ASIA分級(jí):A組獲A級(jí)3例,B級(jí)7例,C級(jí)10例,D級(jí)14例,E級(jí)4例;B組獲A級(jí)3例,B級(jí)6例,C級(jí)16例,D級(jí)17例,E級(jí)12例,兩組與治療前比較均具有顯著性差異(P0.05)。A組發(fā)生消化道出血1例,肺部感染2例,褥瘡2例,下肢深靜脈血栓1例;B組發(fā)生傷口感染1例,硬膜外血腫2例,褥瘡1例,硬膜撕裂導(dǎo)致腦脊液漏1例,兩組并發(fā)癥發(fā)生率比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:與非手術(shù)方法相比,脊髓減壓內(nèi)固定術(shù)可更快、更好的促進(jìn)無骨折脫位型脊髓損傷患者神經(jīng)功能的恢復(fù),且手術(shù)治療不會(huì)增加圍手術(shù)期并發(fā)癥的發(fā)生率。第三部分前、后路減壓內(nèi)固定術(shù)治療無骨折脫位型脊髓損傷療效對(duì)比分析目的:目前國(guó)內(nèi)外文獻(xiàn)通過不同方法和側(cè)面比較均已證實(shí)無骨折脫位型脊髓損傷(CSCIWFD)手術(shù)治療效果要明顯優(yōu)于各種非手術(shù)療法。因此,臨床上一旦遇到CSCIWFD患者出現(xiàn)不同程度的神經(jīng)功能受損時(shí),就需要采取積極的、合適的手術(shù)入路及減壓方法來重建或恢復(fù)椎管的通暢性。用于治療CSCIWFD的手術(shù)方法很多,常見的有頸椎前路椎間盤摘除植骨內(nèi)固定術(shù)、頸前路椎體次全切除植骨內(nèi)固定術(shù)、頸后路單開門椎管擴(kuò)大成形內(nèi)固定術(shù)、頸后路全椎板減壓側(cè)塊螺釘內(nèi)固定術(shù)等。根據(jù)手術(shù)入路劃分,上述術(shù)式可分為經(jīng)前路和后路脊髓減壓術(shù),那么前、后路手術(shù)在總體療效上是否存在差異呢,本研究針對(duì)這一問題進(jìn)行了分組比較。方法:回顧性分析2013年09月~2015年12月在我院接受手術(shù)治療的82例CSCIWFD患者的臨床資料,根據(jù)患者接受的是前路減壓還是后路減壓將其分為兩組:A組(前路組),45例,其中男26例,女19例,年齡22~62歲,平均41.6歲,均接受頸前路減壓內(nèi)固定術(shù)治療;B組(后路組),37例,其中男24例,女13例,年齡19~64歲,平均42.9歲,均接受頸后路減壓內(nèi)固定術(shù)治療。分別記錄兩組患者的手術(shù)時(shí)間、術(shù)中出血量及并發(fā)癥發(fā)生情況;術(shù)前及隨訪過程中以日本骨科協(xié)會(huì)(Japanese Orthopaedic Association,JOA)脊髓損害評(píng)分表(17分法)評(píng)估治療效果;末次隨訪時(shí)根據(jù)ASIA脊髓損傷分級(jí)標(biāo)準(zhǔn)對(duì)兩組患者神經(jīng)功能恢復(fù)恢復(fù)情況進(jìn)行比較分析。結(jié)果:兩組患者均順利接受手術(shù),A組平均手術(shù)時(shí)間(62.6±13.7)min,平均出血量(105.1±16.3)ml;B組手術(shù)時(shí)間(117.9±22.6)min,術(shù)中出血量(240.8±46.3)ml,組間比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。A組JOA評(píng)分由治療前的6.8±1.5分提高至術(shù)后2周時(shí)9.5±1.9分、3個(gè)月時(shí)的11.6±2.6分及末次隨訪時(shí)的13.5±3.1分,治療前后比較差異具有顯著性(P0.05),神經(jīng)功能改善率為65.6%±8.3%,手術(shù)前后比較差異具有顯著性(P0.05);B組JOA評(píng)分由術(shù)前的6.6±1.4分提高至術(shù)后2周時(shí)8.6±1.7分、術(shù)后3個(gè)月時(shí)的10.9±2.5分及末次隨訪時(shí)的13.3±3.4分,手術(shù)前后比較具有顯著統(tǒng)計(jì)學(xué)意義(P0.05),神經(jīng)功能改善率為64.5%±8.5%。術(shù)后2周時(shí)兩組JOA評(píng)分比較具有顯著性差異(P0.05)。但術(shù)后3個(gè)月及末次隨訪時(shí)兩組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。末次隨訪時(shí)根據(jù)ASIA分級(jí):A組獲A級(jí)1例,B級(jí)6例,C級(jí)11例,D級(jí)18例,E級(jí)9例;B組獲A級(jí)1例,B級(jí)4例,C級(jí)11例,D級(jí)13例,E級(jí)8例,與治療前比較均具有顯著性差異(P0.05),術(shù)后組間比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。A組發(fā)生腦脊液漏2例,喉返神經(jīng)牽拉損傷1例;鈦網(wǎng)下沉1例;B組發(fā)生傷口感染1例,液化壞死1例,硬膜外血腫2例,兩組并發(fā)癥發(fā)生率比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:經(jīng)前路和后路脊髓減壓內(nèi)固定術(shù)均可以有效促進(jìn)患者神經(jīng)功能的恢復(fù),圍手術(shù)期并發(fā)癥的發(fā)生率基本相同,但頸前路手術(shù)創(chuàng)傷小,手術(shù)時(shí)間短,術(shù)中出血少,術(shù)前應(yīng)根據(jù)患者脊髓受壓節(jié)段、壓迫源及髓內(nèi)信號(hào)長(zhǎng)度來進(jìn)行科學(xué)、合理的術(shù)式選擇。
[Abstract]:The first part of umbilical cord mesenchymal stem cells to differentiate into neural cells and Study on repair of spinal cord injury to spinal cord injury (spinal cord, injury, SCI) is a spinal injury induced by traumatic disease, appears to be felt in the following segmental injury, motor and autonomic dysfunction. Foreign epidemiological survey, every year around the world there are 13 million new cases of patients with spinal cord injury, and has more than 2 million 500 thousand patient is suffering from varying degrees of sequelae of spinal cord injury problems, and these patients with SCI annual medical expenditures will exceed $6 billion, resulting in a heavy burden to family and society. The current clinical treatment for SCI mainly confined to medicine, surgery and physical intervention. Stem cells for the treatment of SCI is still in the animal related basic research level, such as bone marrow stromal stem cell transplantation, transplantation of olfactory ensheathing cells, umbilical cord blood mesenchymal stem cells Transplantation of umbilical cord derived mesenchymal stem cells (mesenchymal stem cells, MSCs) Nogo transplantation, A vaccine transplantation. Because human umbilical cord mesenchymal stem cells have a wide range of sources, easy amplification, no ethical restrictions, has become a new focus and new direction for the treatment of spinal cord injury in experiments. Umbilical cord blood mesenchymal stem cells in specific environment induction can differentiate into tissue cells with different functions, and promote the protection of factors, and with the integration, regulation, migration and secretion function. When the differentiation of neural cells can promote repair of nerve tissue, improve the neurological function of rats with spinal cord injury. Some scholars in China by transplantation of human umbilical cord blood stem cells for treatment of spinal cord injury in rats, although obtained satisfactory curative effect, but the presence of stem cell differentiation efficiency is low. The purpose of this study is to improve the umbilical cord mesenchymal stem In vitro differentiation efficiency of nerve cells, and transplanted into the spinal cord injury animal model for spinal cord injury in mice, the refractory disease provides a theoretical basis. Methods: collecting and using human umbilical cord explant, collagenase and collagenase and trypsin digestion method combined with the separation of human umbilical cord mesenchymal stem (Human umbilical cord mesenchymal stem cell cells, H UCMSCs), cultured, amplified, Nurr-1 was transfected into human umbilical cord mesenchymal stem cells, and combined with cocktail (ATRA, GGF-2, B factor FGF, PDGF and forskolin) of umbilical cord mesenchymal stem cells to differentiate into neural cells, neural cell marker expression detection by using immunofluorescence technique. The establishment of the model of spinal cord injury in Kunming mice, and differentiated cells were transplanted into spinal cord injury in mice in vivo, immunohistochemical detection of colonization after cell transplantation, judgment The effect of the treatment. Results: human umbilical cord mesenchymal stem cells after induction, cells began to shrink and turned round, and then become stretched into angle, in the process of sustained growth, cells began to appear like drawing synapse like structure of nerve cells. After 2 weeks of induction, by immunofluorescence method can detect the existence of MAP-2 and MBP markers specific nerve cells, and the expression of Nurr-1 of human umbilical cord mesenchymal stem cells differentiation into neural cells. The highest efficiency mice model of spinal cord injury while the destruction of the structure in the gray matter more neuron death, anterior horn neurons surviving, some neurons in nuclei pyknotic.H after transplantation of UCMSCs cells in the injury site proliferated under fluorescence microscope to detect anti human nuclear positive cells, postoperative BBB score of mice increased gradually, hindlimb function gradually improved. Conclusion: Nu RR-1 gene transfection of human umbilical cord mesenchymal stem cells can be obtained in the induction of differentiation and proliferation of the best cocktail factor, can be transplanted into the injured spinal cord injury and proliferation in mice, the hind limb function was significantly improved, showed that the curative effect in the treatment of spinal cord injury without fracture exactly. The second part to a comparative study of the clinical effect of surgical treatment for spinal cord injury dislocation and non operation: spinal cord injury without radiographic abnormality (cervical spinal cord injury without fracture or dislocation, CSCIWFD) is a special type of spinal cord injury, in clinic, usually refers to the neck by minor damage after imaging, such as X-ray, cervical fracture or dislocation were obvious no CT scan, but a kind of Department of orthopedics disease symptoms of spinal cord injury is relatively heavy, accounting for about 23%. of the total number of cervical spinal cord injury consulting literature, CSC The treatment of IWFD is mainly divided into two types, one is the use of glucocorticoid, conservative treatment of neurotrophic drugs and intravenous hyperbaric oxygen treatment (non-surgical), another kind is the surgical treatment of active decompression of spinal cord by surgical means. Each treatment method can promote CSCIWFD patients recover a certain degree of nerve function, but surgical intervention as an invasive operation, the overall effect is better than that of non surgical treatment? To solve this problem, we conducted a clinical comparative study deeply. Methods: a retrospective analysis of October 2013 September ~2015 year in outpatient and inpatient treatment of 110 cases of CSCIWFD patients the clinical data of 92 cases received more than 18 months of clinical follow-up, according to the treatment methods, patients were divided into two groups: A group (non operation group), 38 cases, male 23 cases, female 15 cases, age 19~63 Years old, average 42.1 years old, underwent non operative treatment; group B (operation group), 54 cases, male 35 cases, female 19 cases, age 17~62 years old, average 40.8 years old, underwent surgical treatment. The operation time records of the surgical treatment of patients, intraoperative blood loss and complications in two groups; respectively before treatment, 1 weeks after treatment, 1 months after treatment and at the end of the follow-up by the Japanese Association for Department of orthopedics (Japanese Orthopaedic Association, JOA) spinal cord injury score (17 points) to evaluate the therapeutic effect; at the last follow-up according to the two groups of patients with ASIA spinal cord injury classification results: comparative analysis. All of the patients in group B underwent surgery, including 10 patients underwent anterior cervical discectomy and internal fixation, 17 patients underwent anterior cervical corpectomy and internal fixation, 16 patients underwent posterior open-door laminoplasty with titanium plate fixation, 11 cases of patients undergoing carotid 鍚庤礬鍏ㄦ�庢澘鍑忓帇渚у潡铻洪拤鍥哄畾鏈�.騫沖潎鎵嬫湳鏃墮棿175.2 min,騫沖潎鍑?guó)櫋�閲忎負(fù)210.6 ml.A緇凧OA璇勫垎鐢辨不鐤楀墠鐨，

本文編號(hào)：1393805