转染双耐药基因小鼠骨髓细胞对放化疗的作用

[目的]探讨人突变的胸腺嘧啶合成酶(mTS)及双突变的二氢叶酸还原酶（dmDHFR)基因转染给正常小鼠骨髓细胞后，体内和体外是否对小鼠骨髓细胞具有耐受大剂量放疗与化疗的作用。[方法]将mTS及dmDHFR基因通过反转录病毒转染给小鼠骨髓细胞。再将转染后的骨髓细胞输给经致死剂量照射(9Gy)的小鼠，4周后给小鼠大剂量腹腔注射5-氟尿嘧啶(5—Fu，50mg/kg.d，共5天)和氨甲喋呤(MTX)，观察小鼠骨髓造血集落CFU—S、白细胞、血小板和存活率。[结果]在骨髓移植后第5周，对照组7只小鼠全部死亡，含单药耐药基因TS组有5只存活，含双耐药基因组6只存活。骨髓移植后第8周，再给小鼠腹腔注射大剂量5—Fu(75mg/kg.d，共3天)和MTX(单次300mg/kg)，结果单耐药基因组动物全部死亡，双耐药基因组6只动物全部存活，随着时间推移，存活动物的骨髓功能逐渐恢复，白细胞、血小板逐渐升高。骨髓细胞中有耐药克隆(CFU—GM)形成，骨髓细胞和CFU—S中有相应耐药基因表达。[结论]含有双耐药基因的骨髓细胞对致死剂量照射加5—Fu和MTX处理的小鼠有明显的保护作用。     

双突变二氢叶酸还原酶基因对小鼠的化疗保护作用

目的 探讨人双突变的二氢叶酸还原酶（DHFR）基因对小鼠化疗保护作用。方法 以反转录病毒为载体,将DHFR基因转染入小鼠骨髓干细胞,观察氨甲喋呤（MTX）处理后的骨髓细胞中粒细胞-巨噬细胞克隆形成单位（CFU-GM）的生成情况;观察大剂量MTX化疗后转基因小鼠血象、体重及生存率的变化;用RT-PCR检测转基因小鼠骨髓细胞耐药基因的表达。结果 转染SFG-F／S-NeoR耐药基因的骨髓细胞有耐药克隆的形成,供体小鼠为15.8％,受体小鼠为18．0％,对照组为0;大剂量化疗后,含耐药基因组小鼠血象、体重逐渐恢复正常,生存率为83．3％（第40天）,对照组为0;转基因小鼠骨髓细胞经RT-PCR检测,显示有F／S基因条带（400bp）。结论 DHFR耐药基因可导入小鼠骨髓细胞并获得表达,提高了骨髓细胞对MTX的耐药性。     

逆转录病毒介导的CD/5-FC对胰腺癌细胞的杀伤作用

目的 :探讨大肠埃希菌胞嘧啶脱氨基酶 (CD) /5 -氟胞嘧啶 ( 5 FC)系统对胰腺癌细胞的体外生长抑制作用。方法 :将含CD基因的重组逆转录病毒载体导入胰腺癌细胞形成转化细胞系 ,对转化细胞进行体外药物敏感实验 ,包括 :1)转化细胞在前体药物 5 FC作用下的细胞生长抑制率 ;2 )MTT法检测旁观者效应。结果 :体外实验 5 FC的有效浓度 >2mmol/L时 ,就表现出杀伤作用 ,当 5 FC的有效浓度 >6mmol/L时 ,几乎未见细胞生长 ,PA3 17/CD与TD2混育比例在 90 %时 ,杀伤作用最大 ,相同药物浓度下 ,混育 96h杀伤作用明显高于 2 4h。结论 :CD/5 FC系统对胰腺癌细胞系细胞具有实验性基因治疗作用     

转染人突变dhfr基因的小鼠骨髓细胞对小鼠造血功能的？…

目的：将转染了人突变ｄｈｆｒ基因的第二代小鼠骨髓,移植给经致死剂量照射的第三代小鼠,观察该基因对小鼠造血功能的长期保护作用。方法：分离存活的第二代小鼠骨髓有核细胞,直接移植给经致死剂量照射的同系小鼠,以ＭＴＸ筛选,观察小鼠血象和生存率变化,ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹杂交分析目的基因在小鼠染色体ＤＮＡ中的整合与表达情况。结果：在大剂量ＭＴＸ筛选下,实验组小鼠造血功能逐渐恢复,对照组３周内全部死亡。     

逆转录病毒载体介导MDR1基因转染人外周血造血干细胞的研究

目的：多药耐药基因（ＭＤＲ１）的异常扩增和过度表达是肿瘤细胞产生多药抗药性的重要原因之一。本研究试图将ＭＤＲ１导入骨髓或外周血造知干细胞并使入获得耐药表型,以用于减轻大剂量化疗对造血细胞的毒性。方法：应用反转录病毒载体ｐＨａＭＤＲ１／Ａ将ＭＤＲ１阳性。ＰＣＲ法检测转染细胞所 ＣＦＵ－ＧＭ落集中,外源ＭＤＲ１阳性比例为１／４;而未转染细胞所形成的ＣＦＵ－ＧＭ集匀为阴性。集落形成试验证明,转染和非转染     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的：观察逆转录病毒介导的双自杀基因对K562细胞的杀伤作用，探讨慢性粒细胞白血病的基因治疗方法。方法：通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317，经G418筛选后大量培养产病毒的阳性克隆PA317／CD tk细胞株，收集病毒上清，浓缩后转染K562细胞，再次经G418筛选，获得稳定表达双自杀基因的K562／(：D tk细胞株。用RTl_PCR检测双自杀基因的表达。给予前体药物5-氟胞嘧啶(5-flourocytosine，5-FC)和／或无环乌苷(Ganciciovir，GCv)后MTT法测定转基因组及未转基因组K562细胞的存活率。结果：双自杀基因在K562细胞中可稳定表达，联合使用5-FC和GCv对细胞增殖的杀伤作用及旁杀伤效应高于单独使用5-FC或GCv。结论：逆转录病毒介导自杀基因可有效杀死K562细胞，双自杀基因较单一自杀基因具有更强的抗肿瘤作用。     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用     

多药耐药基因增强骨髓细胞对抗癌药物毒性的抵御能力

目的　探讨多药耐药基因 (mdr1基因 )的骨髓细胞转染增强骨髓细胞对抗癌药的抵御能力。方法　我们用mdr1基因表达质粒pHaMDR1/A ,通过脂质体介导 ,转染小鼠和人骨髓细胞 ,并采用骨髓移植建立小鼠动物模型。结果　成功的将mdr1基因导入了小鼠和人骨髓细胞并获得了表达 ,其细胞已对阿霉素、足叶乙甙 (Vp 16 )、长春新碱和秋水仙碱等产生了明显的抗性。同时 ,转染mdr1基因小鼠骨髓细胞的移植 ,在受体小鼠重建造血功能 ,受体小鼠骨髓细胞亦对抗癌药具有抗性。结论　从小鼠骨髓细胞、人骨髓细胞和小鼠体内模型 3个水平证实了mdr1基因的骨髓细胞转染可增强骨髓细胞对抗癌药的抵御能力     

二氢叶酸还原酶耐药基因对人外周血CD34+细胞的保护作用

目的 探讨突变的二氢叶酸还原酶（ｍＤＨＦＲ）耐药基因在人外周血ＣＤ３４＾＋细胞中抗甲氨喋呤（ＭＴＸ）的效应。方法 应用免疫磁珠分选系统（ＭＡＣＳ）分离纯化外周血ＣＤ３４＾＋细胞后,用含ｍＤ－ＨＦＲ基因的逆转录病毒上清转染人外周血ＣＤ３４＾＋细胞,采用造血祖细胞集落形成实验进行转导后细胞抗ＭＴＸ分析。结果 在ＭＴＸ（２０ｎｍｏｌ／Ｌ）存在的条件下,转导ｍＤＨＦＲ耐药基因的外周血ＣＤ３４＾＋细胞集落形     

胞苷脱氨酶基因对小鼠大剂量化疗的保护作用

Objective:To explore the feasibility of transfecting cytidine deaminase(CD)gene into mouse bone marrow cells in order to observe the drug resistance of high dose Ara-C and improve the tolerance of myelosuppression following combination chemotherapy.Methods:Human cytidine deaminase gene was transfected into mice bone marrow cells by retroviral vector.Resistant colony-forming unit granulocyte-macrophage(CFU-GM)assay was performed after the transfected mice bone marrow cells treated by the Ara-C.DNA was extracted from mice bone marrow cells.The drug resistant gene in mice bone marrow cells after transfection was detected by PCR.Results:Bone marrow cells of lhe donor mice cultured with lhe retroviral producer cells showed the drug resistant colonies and resistance to Ara-C,so did accept mice transplanted with the CD gene(CFU-GM of donor mice was 52%,X2=124.62,P＜0.01:accept mice was 54%,X2=126.26.P＜0.01,both compared with the contrast group).The animal survival rate was significantly higher in gene transfected group than that of the control(X2=7.42.P＜0.01).CD gene of transfected bone marrow cells was confirmed by PCR.Conclusion:CD gene can be transfected into bone marrow cells of mice efficiently and increase the drug resistance to Ara-C.     

IL-3基因在γ线照后小鼠骨髓造血细胞中表达的形态定量研究

本文用图像分析和免疫组化等技术研究小鼠骨髓辐射损伤后造血细胞IL- 3 基因表达及意义。结果表明: 照后4 周内小鼠骨髓出现明显损伤及损伤后重建现象。免疫组化显示正常小鼠骨髓中仅少部分造血细胞浆内IL- 3 呈弱阳性; 5-5Gy 照后6h ～6d, 造血细胞浆内IL- 3 进行性减少, 尤其照射1d 后, 基本不见造血细胞表达IL- 3 ; 照射后10d , 少部分造血细胞中始见IL- 3 表达, 呈弱阳性; 照射后15 ～21d, 造血细胞中IL- 3 逐渐增加, 达最高峰, 表现为IL-3 表达的造血细胞增多, 阳性强度增强。然后4 周, 造血细胞内IL- 3 呈减少趋势, 但仍高于正常小鼠骨髓。定量分析表明IL- 3 于照后15 ～28d , 其单位面积的积分光密度及数密度明显增加( P< 0-01 或P< 0-05) , 尤以照后21d 为显著( P< 0-01) 。据此作者认为, 内源性IL- 3 基因表达在骨髓辐射损伤后造血功能重建中可能起明显的促进作用     

Distribution of preleukemic cells in fractionated bone marrow of mice

Separation of preleukemic cells (having the potential to develop further into overt T-cell leukemias) from bone marrow cell populations was attempted. Donor mice of preleukemic bone marrow included C57BL/6 mice inoculated intrathymically with D-RadLV or AKR/J mice carrying spontaneous preleukemic cells among their bone marrow cells. Fractionation of bone marrow cells suspended in bovine serum albumin (BSA) by equilibrium density centrifugation or by velocity sedimentation at 1 g unit gravity using discontinuous density gradient of Ficoll was applied. The leukemogenic potential of the separated bone marrow fractions was tested by evaluating leukemia development following their transfer into appropriate recipients. No enrichment of D-RadLV-induced preleukemic cells was found in any of the four bone marrow fractions obtained following separation on BSA gradient. Separation of D-RadLV-induced preleukemic cells was afforded by using the Ficoll gradients. Preleukemic cells were located mainly among four out of 21 fractions tested, consisting mostly of 10 to 14 μm size cells. In contrast, preleukemic cells from AKR donors were distributed among most of the separated fractions. It is suggested that these variable results may reflect homogeneity or heterogeneity of progenitor target cells undergoing transformation.     

粒细胞集落刺激因子减轻混合骨髓移植后移植物抗宿主病的实验研究

Objective: How to reduce the incidence and severity of acute graft-versus-host disease (aGVHD) is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (allo-BMT). The low incidence of severe aGVHD observed in allogeneic peripheral blood stem cell transplantation (allo-PBSCT), which may be related to modulating immune function of T lymphocytes by granulocyte colony-stimulating factor (G-CSF) primed donors. The study aimed to explore whether aGVHD could be alleviated by syngeneic bone marrow mixed with G-CSF-mobilized H-2 haploidentical marrow grafting. Methods: Female BALB/c mice and neonatal BALB/c mice were recipients and male (BALB/c × C57BL/6)F1(BCF1) mice were donor mice respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g body weight or saline for 6 days, and splenocytes were harvested on day 6. Spleen index (SI) represented GVHD in neonatal mice after the intraperitoneal injection of mixed spleen cells. Lethally irradiated (60Co, 8.5 Gy) adult mice were transplanted with a mixture of syngeneic plus G-CSF-mobilized (control diluents) H-2 haploidentical marrow cells. Survival time and survival rate of the recipients were observed after mixed marrow transplantation (MBMT). GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2, IL-4 and INF-γ). Fluorescence-activated cell sorting (FACS) analysis was used to detect T cells phenotype. Results: (1) The neonatal mice subject to injection of 2:1 and 1:1 mixed spleen cells and H-2 haploidentical spleen cells all suffered from aGVHD. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes was dramatically reduced. (2) The aGVHD signs and histological change were observed in most mice of 2:1 and 1:1 MBMT groups. However, the survival time of G-CSF-mobilized MBMT was longer than in control groups and these mice had signs of moderate GVHD. (3) L3T4 cells and relative ratio in both subsets was significantly reduced in G-CSF-treated donor mice. The total number of Thy1.2 and lyt2 cells was increased after G-CSF pretreatment of donors, but no statistical difference. (4) The supernatants from a primary MLR were collected at 48 h for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and INF-γ after stimulating with concanavalin A for 48 h. Conclusion: The GVHD could be reduced using syngeneic bone marrow mixed with H-2 haploidentical marrow cells. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes or marrow cells could be further moderated, which is associated with increased IL-4 production and decreased IL-2 and INF-γ production.     

双突变二氢叶酸还原酶基因对小鼠骨髓细胞化疗保护作用的体外研究

目的探讨将人双突变的二氢叶酸还原酶基因(dihydrofolate reductase,DHFR)导入小鼠骨髓细胞中,观察小鼠骨髓细胞对大剂量甲氨蝶呤(methotrexate,MTX)的耐受性,研究骨髓耐受大剂量化疗的可行性。方法以反转录病毒为载体,将DHFR通过共培养转染入小鼠骨髓干细胞,观察共培养后的骨髓细胞耐MTX CFU-GM生成情况;小鼠骨髓细胞提取的DNA,用PCR检测小鼠骨髓细胞耐药基因的表达。结果含有耐药基因(SFG-F/S)的骨髓细胞耐药克隆为16%,对照组为0,χ2=47.88,P<0.005;转基因小鼠骨髓细胞经PCR检测,显示有F/S基因条带;耐药基因转染后小鼠骨髓细胞对MTX的耐受明显增加。结论耐药基因可以进入小鼠骨髓细胞并且获得共表达,提高了造血细胞对MTX的耐药性。     

Study of bone marrow cells in non-Hodgkin's lymphoma by DNA analysis

Using Southern-blot analysis, we studied samples of bone marrow (BM) cells from 73 patients with non-Hodgkin's lymphoma (NHL) in various clinical status. The frequency of gene rearrangement was disease-status dependent with a frequency of 65.8% at the diagnostic stage, 81.8% after relapse and 33.3% upon complete remission (CR). BM involvement was evident in a substantial portion of patients with untreated and relapsed lymphoma. The significance of BM involvement by DNA hybridization in relation to conventional clinical staging and histological grade was studied. By Southern-blot analysis, BM involvement was found in 76% of the patients at clinical stages (CS) I–III. The incidence of BM involvement in low, intermediate and high grades of NHL (Working Formulation) was 57%(4/7), 67%(22/33), and 89%(8/9) respectively. A comparative study of conventional BM biopsy vs DNA hybridization in a group of 47 NHL patients showed that all 12 patients (100%) with morphological BM involvement and 25 out of 35 patients (71%) with morphologically normal BM had clonal rearrangements of immunoglobulin (Ig), heavy chain and/or light chain, or T-cell receptor β chain (TCRβ) genes in BM cells. The false negative rate in conventional BM biopsy was 53%(25/47). Southern-blot analysis on lymph nodes (LN) and BM cells from 37 patients showed that 6 patients (16%) had cross-lineage or different rearranged patterns in the same or different tissues. Southern-blot analysis was found to be highly reliable for the detection of even minimal populations of lymphoma cells in the BM and therefore should be the diagnostic choice for clinical staging of lymphoma.     

粒细胞集落刺激因子对骨髓移植及外周血干细胞保护的骨髓造血重建

研究粒细胞集落刺激因子对骨髓移植及外周血造血干细胞保护患者的骨髓造血重建中的作用。方法；采用自体／异基因骨髓移植和外周血造血祖细胞保护的大剂量化疗，对１１例急性白血病及中－高恶性度非何杰林巴瘤患者进行治疗。     

乳腺癌骨髓微小转移灶的检测与意义

骨是乳腺癌最常见的转移部位之一 ,大约 3 0 %左右的可手术乳腺癌存在骨髓微小转移灶。主要阐述了乳腺癌骨髓微小转移灶的检测方法及其预后意义 ,同时对乳腺癌微小转移灶的生物学特性和对辅助治疗的反应加以综述