本文關(guān)鍵詞：環(huán)磷酰胺在移植治療再生障礙性貧血中的應(yīng)用及對造血功能的影響　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 環(huán)磷酰胺 造血干細胞移植 移植物抗宿主病 動物模型 造血功能

【摘要】：研究背景及目的背景再生障礙性貧血(aplastic anemia,AA)是一組由理化因素、生物因素及不明原因引起的骨髓造血衰竭性疾病,具有致死率高、預(yù)后差等特點,造血干細胞移植成為根治AA的最有效方法。然而由于計劃生育政策,同胞兄妹供者較少,對于單倍型造血干細胞移植來說,患者父母及兄弟姐妹均可作為其供者來源,使得單倍型造血干細胞移植成為主流力量。但嚴重的移植物抗宿主病(graft versus host disease,GVHD)和移植物排斥反應(yīng)的發(fā)生成為主要障礙,直接影響移植后的長期生存率。環(huán)磷酰胺(CTX)具有強效的免疫抑制抗腫瘤作用,移植后高劑量CTX誘導(dǎo)免疫耐受在單倍型移植中的應(yīng)用引起關(guān)注,但是應(yīng)用時機和劑量很重要。CTX誘導(dǎo)耐受新方法在單倍型移植中占有重要作用,但對于CTX誘導(dǎo)免疫耐受的作用機制及特點目前尚未完全清楚,特別擔心對造血重建的影響,本研究通過小鼠實體實驗觀察CTX對造血干細胞的影響,為移植工作提供理論依據(jù)。目的1.評價單倍型移植后應(yīng)用高劑量CTX誘導(dǎo)免疫耐受治療重型再生障礙性貧血(SAA)的臨床療效;2.通過動物實驗觀察不同劑量CTX對小鼠造血功能的影響。研究方法1.2014年1月至2015年8月期間單中心中國人民解放軍陸軍總醫(yī)院血液科移植后使用高劑量CTX誘導(dǎo)免疫耐受單倍型造血干細胞移植新方法治療的16例SAA患者,男7例,女9例,患者中位年齡14.5(4~30)歲。所有均為親緣單倍型供者,其中父親供者9例,母親供者5例,同胞兄妹供者2例,供者中位年齡31(20~45)歲。觀察全部患者移植后植入情況、造血重建(包括中性粒細胞及血小板植活時間)、移植相關(guān)并發(fā)癥(移植物抗宿主病、感染及出血)、存活情況及隨訪時間等指標。2.選取雌性純系BALB/c小鼠,按照常規(guī)方法制成再生障礙性貧血小鼠模型,所有造模成功后的小鼠隨機分組以供實驗需要。將環(huán)磷酰胺藥物以無菌生理鹽水溶解,按CTX處理劑量(380mg/kg、200mg/kg、100mg/kg)作用于實驗組小鼠,動態(tài)觀察實驗組(HD-CTX組、MD-CTX組、LD-CTX組)、AA模型對照組、正常對照組小鼠一般狀況、外周血血常規(guī)變化、雙側(cè)股骨有核細胞數(shù)(NC)及股骨骨髓病理切片、半固體甲基纖維素培養(yǎng)法測定紅系集落形成單位(BFU-E)、粒單系集落形成單位(CFU-GM)及流式細胞術(shù)檢測外周血及骨髓CD34+細胞變化。結(jié)果1.全部16例患者除1例原發(fā)排斥,余15例完全植入,其中有2例患者第1次未植入,行二次移植達到完全供者植入狀態(tài)。粒細胞植活中位時間15.5(14~26)天、血小板植活中位時間22.0(17~32)天。隨訪至2016年8月,中位隨訪時間17.4(9~28)月,存活患者隨訪時間均在6個月以上,最長隨訪時間達28個月。移植后共有8例發(fā)生急性GVHD,其中IV度急性GVHD 2例,III度急性GVHD 3例,I~II度急性GVHD 3例,按部位分為腸道3例、肝臟3例、皮膚2例;2例發(fā)生慢性GVHD,均發(fā)生在皮膚。肺部感染7例;巨細胞病毒感染3例,其中2例發(fā)展為巨細胞肺炎,予更昔洛韋、磷甲酸鈉治療后好轉(zhuǎn),無肝靜脈綜合征及移植后淋巴細胞增殖發(fā)生;出血性膀胱炎4例。所有患者1例因未植入死亡,1例因嚴重肺部感染死亡,1例因急性GVHD死亡,其余13例仍存活。2.1)全部受鼠基于AA再障發(fā)病機制建造AA小鼠模型成功。2)正常對照組白細胞及血小板數(shù)目波動在正常范圍;AA模型組白細胞及血小板變化大致相似,均進行性下降,第5天開始明顯下降,分別為(0.46±0.32)×109/L、(33.4±14.67)×109/L,差異均有統(tǒng)計學意義(P0.05);LD-CTX及MD-CTX組白細胞數(shù)目在第9天開始回升。HD-CTX組白細胞數(shù)目變化與模型組大致相同。AA模型組骨髓及外周血CD34+細胞進行性下降,第5天降至較低水平,(1.76±0.28)×109/L,第7天開始回升,但仍低于正常,為(2.86±0.45)×109/L,差異有統(tǒng)計學意義(P0.05)。LD-CTX及MD-CTX組第3天骨髓及外周血CD34+細胞降至最低,分別(0.66±0.30)×109/L、(0.72±0.40)×109/L,差異有統(tǒng)計學意義(P0.01),第7天細胞數(shù)開始回升。HD-CTX組骨髓及外周血CD34+細胞第3天最低,為(0.49±0.38)×109/L,隨后一直處于低水平,差異有統(tǒng)計學意義(P0.05)。模型AA組CFU-GM、CFU-E集落形成數(shù)明顯減少,48h、96h分別形成的CFU-GM、CFU-E集落數(shù)為(10.00±5.00)個、(5.00±3.00)個、(12.67±3.79)個、(5.33±3.21)個,差異均有統(tǒng)計學意義(P0.05)。LD-CTX、MD-CTX、HD-CTX組與模型組比較無明顯差異。結(jié)論1.移植后高劑量CTX誘導(dǎo)免疫耐受單倍型異基因造血干細胞移植治療重型再生障礙性貧血,造血重建穩(wěn)定,有較好的臨床療效。2.單倍型造血干細胞移植后應(yīng)用高劑量CTX誘導(dǎo)免疫耐受可減少移植相關(guān)并發(fā)癥,是預(yù)防單倍型移植GVHD發(fā)生的新方法。3.將受鼠BALB/C小鼠經(jīng)Co60γ放射線亞致死量(5.5Gy)全身照射,將DBA/2小鼠的淋巴細胞懸液在4小時內(nèi)經(jīng)尾靜脈輸注受鼠(BALB/C鼠)體內(nèi),該過程大致模擬AA發(fā)病過程,制造AA小鼠模型,觀察AA小鼠外周血血常規(guī)變化、骨髓有核細胞數(shù)及骨髓病理變化均符合AA的病理變化,說明小鼠AA造模成功。4.動物實驗證實CTX對骨髓造血功能無明顯損傷作用,為臨床移植工作應(yīng)用高劑量CTX誘導(dǎo)免疫耐受提供理論依據(jù)。
[Abstract]:Background and objective background of aplastic anemia (aplastic anemia AA) is a group of physical and chemical factors, biological factors and unexplained bone marrow failure disease, with high mortality and poor prognosis, hematopoietic stem cell transplantation has become the most effective method to cure AA. However, because of the one-child policy sibling donor, less for haploidentical hematopoietic stem cell transplantation for patients with parents and siblings can be used as the donor, the haploidentical hematopoietic stem cell transplantation has become main force. But severe graft versus host disease (graft versus host disease, GVHD) and graft rejection has become main obstacle and directly influence the long-term survival rate after transplantation. Cyclophosphamide (CTX) immune suppression has potent antitumor effect, application of high dose of CTX after transplantation induced immune tolerance in transplantation induced by haplotype Now, but the timing and dose of.CTX is very important to a new method for inducing tolerance in haplotype transplantation plays an important role, but the mechanism and characteristics of immune tolerance induced by CTX is not fully understood, particularly worried about the impact on hematopoietic reconstruction, the effects on hematopoietic stem cell research by small rat experimental observations of CTX entity, provide the theoretical basis for the transplantation. Objective 1. to evaluate the haplotype after transplantation immune tolerance induced by high dose CTX in treatment of severe aplastic anemia (SAA) clinical curative effect; 2. through animal experiments to observe the effects of different doses of CTX on hematopoietic function in mice. Methods 16 cases of SAA patients 1.2014 years from January to August 2015 during the use of a single center China people the PLA General Hospital Department of Hematology after transplantation immune tolerance induced by high dose CTX haploidentical hematopoietic stem cell transplantation therapy, 7 cases were male, 9 were female, Patients with a median age of 14.5 (4~30) years old. All were haploidentical donor, the father for 9 cases, 5 cases of maternal donors, 2 cases of sibling donor donor, the median age was 31 years (20~45). The implantation was observed in all patients after transplantation, hematopoietic reconstruction (including neutrophils and platelet engraftment time), transplantation related complications (graft-versus-host disease, infection and bleeding), survival rate and follow-up time index.2. female inbred BALB/c mice, according to the conventional method made the model of AA mice, all afterinjecting mice were randomly divided to experiment. The cyclophosphamide drugs with sterile saline solution, treatment dose according to CTX (380mg/kg, 200mg/kg, 100mg/kg) in experimental mice, dynamic observation of the experimental group (HD-CTX group, MD-CTX group, LD-CTX group), AA model group, normal control group of Mice Peripheral Blood 甯歌鍙樺寲,鍙屼晶鑲￠鏈夋牳緇嗚優(yōu)鏁，

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