细胞因子治疗4.5Gyγ射线照射比格犬造血损伤的机制初探

目的 探讨多种细胞因子配伍应用治疗4.5 Gy γ射线照射比格犬造血系统损伤的作用及可能机制。方法 16只比格犬均给予4.5 Gy ⁶⁰Co γ射线全身照射,随机分为照射对照、综合对症和细胞因子3组。细胞因子组在综合对症支持治疗的基础上应用rhG-CSF、rhIL-11和rhIL-2联合治疗,采用流式细胞术检测外周血中CD34+细胞含量、有核细胞周期及凋亡比例。结果 4.5 Gy γ射线照射犬外周血中CD34+细胞含量在照射后1 d明显下降(照射对照组和综合对症组分别为照前值的61.3%和52.1%),G₀/G₁期有核细胞比例增加(分别为99.27%和99.49%),且凋亡率(分别为26.93%和21.29%)和坏死率(分别为3.27%和4.14%)明显升高(与照前值比较, P<0.05);而经过细胞因子治疗后,外周血中CD34+细胞含量在照射后1 d即明显升高(为照前值的135.6%),G₀/G₁期有核细胞比例(99.71%)进一步增加,其凋亡率(5.66%)和坏死率(1.60%)明显低于照射对照和综合对症组。结论 本研究的细胞因子组合可能通过动员骨髓中CD34+细胞到外周血,使细胞周期阻滞在G₀/G₁期,减少细胞凋亡,从而促进极重度骨髓型急性放射病犬造血功能的恢复。     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.     

细胞因子诱导的杀伤细胞研究进展

细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)是将人外周血单个核细胞在体外用多种刺激因子,如抗cD3一McAb、IL．2、血清干扰素-γ(IFN-γ)、IL-1等共同培养一段时间后获得的一群异质性细胞。它兼有T淋巴细胞强大的抗瘤活性和NK细胞的非MHC限制性杀瘤优点,比淋巴因子激活的杀伤细胞(LAK)、CTL、TIL的增殖能力和细胞毒性强,因而成为近10年肿瘤生物治疗的热点。现就CIK细胞的特点、诱导、杀瘤机制及应用前景等综述如下。     

Experimental therapeutic effect of combined cytokines on beagle dogs exposed to 4.5 Gy γ-rays

Objective To evaluate the effects of combined administration of recombinant human interleukin-11(rhIL-11),recombinant human G-CSF(rhG-CSF)and recombinant human interleukin-2 (rhIL-2)on acute radiation sickness(ARS)beagles.Methods Sixteen beagle were irradiated with 4.5 Gy60 Co γ-rays to establish ARS models,and were divided into irradiation control group,supportive care group and combined cytokines treatment group.After irradiation irradiation control group was given no treatment,the dogs in supportive care group received purely symptomatic treatment,while combined cytokines treatment group received rhIL-11 50μg/(kg·d)and rbG-CSF 10μg/(kg·d)subcutaneously(0-14 d)and rhIL-2 1×1 06 U/d(29-43 d)besides symptomatic treatment.Manifestation and characteristics of ARS beagles were observed,and the survival time were recorded.At last,post-mortem examination and histological examination were performed.Results All animals underwent nausea,diarrhea and fever.After irradiation,all animals in irradiation control group died in two weeks,and the mean survival time was 12.7 d,while only one died at 33 d in supportive care group.All dogs in combined cytokine group survived at 45 day after exposure,and their haematopoiesis and gastrointestinal tract were recovered.Conclusions Combination of rhIL-11 + rhG-CSF + rhIL-2 treatment could be significantly effective on ARS beagles irradiated by 4.5 Gy60 Co γ-rays,which could accelerate injured haemotopoiesis and intestinal tract recovery,increase the survival rate and improve the life quality of animals.     

Clotting mechanism in beagles irradiated by 4.5 Gy γ-rays

Objective To explore the clotting mechanism in beagles irradiated by 4.5 Gy γ-rays after treatment with supportive care,or supportive care and combined cytokines.Methods Sixteen beagles were divided into irradiation control group,Supportive care group and combined cytokines treatment group.Platelet aggregation test,thrombelagtography (TEG) and the time measurement were analyzed in vitro.Results In irradiation group and supportive care group,the platelet aggregation rates in beagles were decreased markedly and the k value of TEG was increased 7 d post-irradiation,while those indexes in combined cytokines treatment group changed little.At 14 d post-irradiation,each parameter of TEG in irradiated group changed obviously.The values of r,k,r+k and M were elevated significantly,clotting time and the maximum coagulation time of thrombus delayed,the Ma value was decreased markedly,and the maximum elasticity amplitude of thrombus was diminished.All parameters in combined cytokines treatment group were better than those in supportive care group.The thrombin time was prolonged obviously in irradiated group 14 d post-irradiation,while the thrombin time was the longest at 2-3 weeks post irradiation in supportive care group and combined cytokines treatment group(P>0.05).Conclusions Cytokines could improve the platelet aggregation and the blood clotting functions of beagles suffering from acute radiation sickness.     

人白介素24腺病毒载体的构建与鉴定

目的构建人白介素24（hIL-24）的腺病毒载体,获得hIL-24重组腺病毒子,为hIL-24进行肿瘤的基因治疗奠定基础。方法以pcDNA3．0-hIL-24重组质料为模板,PCR扩增hIL-24,酶切连接到带有GFP标记的pAdTrack—CMVR质粒上,PmeI线性化重组质粒pAdTrack—CMV-hIL-24,与腺病毒质粒pAdEasy-1共转化BJ5183细菌,获得重组腺病毒载体pAdEasy-1-pAdTrack-CMV-hIL-24,经Pacl线性化后转染QBI-293A包装细胞。收获腺病毒重组病毒子,RT-PCR和Western—blotting鉴定。结果测序结果显示hIL-24序列正确,RT—PCR和Western-blotting检测到了hIL-24的表达。结论成功构建人白介素24的重组腺病毒载体pAdEasy-1-pAdTrack,CMV—hIL-24。获得了人白介素24重组病毒子Ad-hIL-24。     

人IL-31基因克隆、表达及在皮肤炎症中的作用

目的 克隆人IL-31基因,构建原核表达载体,并诱导其在大肠杆菌中表达,研究hIL-31蛋白与皮肤炎症的关系.方法 采用RT-PCR克隆人IL-31基因,将其克隆到原核表达载体pET28a( ),并通过IPTG诱导目的基因在大肠杆菌中表达,用该目的蛋白刺激人表皮角质细胞HaCaT,RT-PCR检测趋化因子MIP-3β、TARC及TCA-3(I-309)mRNA的表达;小鼠皮内注射该目的蛋白,观察局部炎症表现,皮肤标本HE染色观察皮肤炎性特征,采血进行白细胞计数及分类,ELISA法检测血清IL-1βIL-6和TNF-α水平,流式细胞仪分析胸腺及脾脏T细胞亚群.结果 成功获得495bp的人IL-31基因,原核表达质粒构建正确,IPTG诱导目的基因在大肠杆菌中大量表达;该目的蛋白可刺激HaCaT表达趋化因子MIP-3β、TARC、I-309;小鼠皮肤注射部位有脱毛,HE染色可见炎性细胞浸润,外周血白细胞总数增加,中性粒细胞比例增高,血清IL-1β、IL-6和TNF-α水平增高,脾脏CD4 T细胞百分比增高,CD8 T细胞百分比减少.结论 人IL-31基因克隆及原核表达已获成功,hIL-31蛋白可刺激HaCaT细胞表达MIP-3β、TARC、I-309,诱导小鼠皮肤炎症反应.     

神经生长因子的临床治疗应用及用药

神经生长因子 (NGF)在临床前试验和临床上用于治疗阿尔茨海默病 (Alzheimer’sdisease,AD )、外周神经病等神经退行性疾病 ,可以改善神经退行性变和神经伤。同时 ,植入、移植等NGF用药方法的研究也不断深入 ,不久有望用于临床。     

细胞因子在急性辐射损伤治疗中的作用

急性辐射损伤以及肿瘤患者在放疗或化疗后,均可发生程度不等的骨髓功能抑制。一些细胞因子通过刺激不同分化时期、不同类型的靶细胞影响其增殖和分化,从而对骨髓造血细胞起辐射防护和促进骨髓造血恢复的作用。随着基因工程的发展,人们可以获得越来越多的高纯度重组造血因子,这些因子在急性辐射损伤的实验研究和临床救治中发挥了极其重要的作用。同时,细胞因子治疗也存在一些问题,有待进一步研究和解决。     

rhIL-18在不同原核表达系统中的表达和活性比较

目的研究rhIL-18包涵体、可溶性（His融合型）两种形式蛋白的生物学活性。方法从已构建的pBV220-IL-18重组质粒中双酶切获取IL-18基因，回收后重组至pLHis质粒构建重组载体。42℃热诱导pBV220-IL-18表达，产生rhIL-18包涵体，IPTG诱导pLHis-IL-18表达，产生可溶性rhIL-18蛋白。两者分别刺激人外周血单个核细胞，ELISA法检测上清中IFN-γ的含量。结果两种重组表达载体均表达产生了rhIL-18，且均可刺激人PMBC上清产生IFN-γ，可溶性rhIL-18刺激人外周血产生IFN-γ的能力高于包涵体形式的rhIL-18。结论可溶性rhIL-18具有比包涵体形式的rhIL-18更强的活性．这为IL-18原核表达基因工程药物的开发提供了新思路。