Objective To investigate the effect of tumor associated glycoprotein-72 (TAG-72) redirected T lymphocytes on breast cancer cells. Methods Peripheral blood mononuclear cells (PBMCs)were isolated from healthy volunteers. The recombinant vector anti-TAG-72-scFv-CD3ζ-pcDNA 3.0 were transfected into PBMCs by lipofectamineTM2000 (transfection group), PBMCs transfected with plasmid pcDNA 3.0 as control group. MCF-7 and Bcap37 cells were cocultivated with PBMCs of transfection group and control group, respectively, and antitumor response of G1 block was observed. Results G1 block rate of MCF-7 cells in transfection group was (82.3±6.9)%, which was significantly higher than that in control group 〔(43.4±3.9)%, P<0.05〕. G1 block rate of Bcap37 cells in transfection group was (51.3±4.7)%, and not differed from that in control group 〔(45.6±2.5)%, P>0.05〕. Conclusion TAG-72 redirected T lymphocytes can inhibit the cell proliferation of TAG-72 positive breast cancer cells, and it may provide valuable tools for the cellular immunotherapy.
The bone marrow mononuclear cell(BMMNC) subset comprises mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells. These cells can differentiate into cardiomyocytes, vascular endothelial cells and smooth muscle cells, and they can also release a wide array of cytokines that exert their effects on surrounding cells, including inducing neovascularization, preventing apoptosis of home cells and homing of endogenous systemic repairing cells. Many trials have been developed to evaluate the effect of bone marrow mononuclear cell transplantation in treating ischemia heart diseases in this country and others. Several routes have been used to deliver these cells to human myocardium or to the coronary circulation in these trials, such as intracoronary injection, intravenous infusion, direct injection into the ventricular wall, or transepicardial/transendocardial infusions,and the cells are constructed into fragmented cell sheets to improve cell retention, or some cytokines are used to enhance therapeutic effect. Although the results of the recent clinical trials in this area are rather conflicting, these therapeutic approaches seem to be promising forthe treatment of ischemic heart disease. In this review, many aspects of bone marrow mononuclear cell transplantation in myocardial infarction are summarized such as the mechanism, delivery routes, retaining of cells, homing, survival and future development, etc.
Abstract: Objective To observe the changes in morphology, structure, and ventricular function of infarct heart after bone marrow mononuclear cells (BMMNC) implantation. Methods Twenty-four dogs were divided into four groups with random number table, acute myocardial infarction (AM I) control group , AM I-BMMNC group , old myocardial infarct ion (OMI) control group and OM I-BMMNC group , 6 dogs each group. Autologous BMMNC were injected into infarct and peri-infarct myocardium fo r transplantation in AM I-BMMNC group and OM I-BMMNC group. The same volume of no-cells phosphate buffered solution (PBS) was injected into the myocardium in AM Icontrol group and OM I-control group. Before and at six weeks of cell t ransplantation, ult rasonic cardiography (UCG) were performed to observe the change of heart morphology and function, then the heart was harvested for morphological and histological study. Results U CG showed that left ventricular end diastolic dimension (LV EDD) , left ventricular end diastolic volume (LVEDV ) , the thickness of left ventricular postwall (LVPW ) in AM I-BMMNC group were significantly less than those in AM I-control group (32. 5±5. 1mm vs. 36. 6±3. 4mm , 46. 7±12. 1m l vs. 57. 5±10. 1m l, 6. 2±0. 6mm vs. 6. 9±0. 9mm; P lt; 0. 05). LVEDD, LVEDV , LVPW in OM I-BMMNC group were significantly less than those in OM I-control group (32. 8±4. 2 mm vs. 36. 8±4. 4mm , 48. 2±12. 9m l vs. 60.6±16.5m l, 7. 0±0. 4mm vs. 7. 3±0. 5mm; P lt; 0. 05). The value of eject fraction (EF) in OM I-BMMNC group were significantly higher than that in OM I-control group (53. 3% ±10. 3% vs. 44. 7%±10. 1% ). Compared with their control group in morphological measurement, the increase of infarct region thickness (7. 0 ± 1. 9mm vs. 5. 0 ±2.0mm , 6.0±0. 6mm vs. 4. 0±0. 5mm; P lt; 0. 05) and the reduction of infarct region length (25. 5±5. 2mm vs. 32. 1±612mm , 33. 6±5. 5mm vs. 39. 0±3. 2mm , P lt; 0. 05) were observed after transplantation in AM I-BMMNC group and OM I-BMMNC group, no ventricular aneurysm was found in AM I-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OM I-BMMNC group (0. 581±0. 013 vs. 0. 566±0.015; P lt; 0. 05). Both in AM I-BMMNC group and OM I-BMMNC group, fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescencew as irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transp lantation both in AMI and in OM I, and significant lymphocyte infiltration in AM I-BMMNC group. Conclusion BMMNC implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and st ructure, and improve neovascularization in histology, and improve the heart function.
Objective To determine the application values of gene chip technique in cardiovascular surgical clinical and research work. Microarray for gene expression profiles was used to screen out the differentially expressed genes during cardiopulmonary bypass(CPB) in peripheral blood mononuclear cell. By doing these, it was hoped that some clues in inflammatory response during CPB could be found out. Methods The patients’ oxygenated bloods were drawn immediately before onset and termination of CPB. Peripheral blood mononuclear cell (PBMC) were obtained from heparinised blood by Ficoll gradient centrifugation. The differentially expression was measured using BD AtlasTM cDNA Expression Arrays. The candidate genes were corroborated by semiquantitative reverse transcriptionpolymerase chain reaction (RT-PCR). Results Gene chip technique was successfully used in CPB study. The gene expression profiles of cytokines of PBMC during CPB were screened out. Interleukin 6 and Wnt5a were the differentially expressed genes. But the validity using semiquantitative RT-PCR found no statistically difference(P=0.888,0.135). Conclusion Microarray technique has positive application values in the study of cytokines during CPB. cDNA microarray for gene expression profiles can primarily screen out differentially expression genes during CPB. These genes may be engaged in inflammation and other pathophysiological reactions during CPB. PBMC is not the major source of cytokines during CPB.
Objective To investigate the effectiveness of autologous bone marrow mononuclear cells transplantation on lower l imb chronic venous ulcer. Methods Between May 2009 and September 2010, 17 patients with lower l imb chronic venous ulcer were treated with autologous bone marrow mononuclear cells transplantation (transplantation group) and 10patients treated without cells transplantation served as control group. In the transplantation group, there were 9 males and 8 females with age of (33.3 ± 6.1) years, including 11 cases of simple great saphenous vein varicosity and 6 cases of chronic venous insufficiency; the area of ulcer was (4.39 ± 2.46) cm2; and the duration of ulcer ranged from 3 months to 6 years. In the control group, there were 4 males and 6 females with age of (39.2 ± 10.3) years, including 7 cases of simple great saphenous vein varicosity and 3 cases of chronic venous insufficiency; and the area of ulcer was (5.51 ± 2.63) cm2; and the duration of ulcer ranged from 3 months to 2 years. All patients in both groups were classified as C6 according to Cl inical Etiology Anatomy Pathophysiology (CEAP) classification. No signficant difference was found in the general data between 2 groups (P gt; 0.05). The heal ing process of ulcer was observed. The granulation tissue was harvested for HE staining before operation and at 3 days after operation in the transplantation group. The microvessel density (MVD) and vascular endothel ial growth factor (VEGF) expression of ulcer granulation tissue were observed. Results In the transplantation group, ulcer heal ing was accelerated; complete heal ing was observed in 15 cases, partial heal ing in 1 case, and no heal ing in 1 case with the median heal ing time of 22 days. However, in the control group, the heal ing process was slower; complete heal ing of ulcer was observed in 7 cases and no heal ing in 3 cases with the median heal ing time of 57.5 days. There was significant difference in the heal ing time between 2 groups (Z=0.001 4, P=0.002 7). HE staining showed a great number of microvessels in the granulation tissue in the transplantation group. The immunohistochemical staining showed that MVD was significantly increased (t=3.120, P=0.008) after cell ransplantation (32.1 ± 12.8) when compared with that before transplantation (22.1 ± 6.7). The VEGF expressionafter transplantation (8.05% ± 5.10%) was increased sl ightly when compared with that before transplantation (6.13% ±4.20%), but the difference was not significant (t=1.150, P=0.268). Conclusion Autologous bone marrow mononuclear cellstransplantation can stimulate granulation tissue growth and improve ulcer heal ing.
Objective To investigate the efficacy of autologous bone marrow mononuclear cells transplantation in treating lower l imb thromboangiitis obl iterans (TAO). Methods From January 2005 to November 2008, 25 patients (27 l imbs) with lower l imb TAO were treated. There were 24 males (26 l imbs) and 1 female (1 l imb), aging 16-44 years (33 years on average). Fifteen left l imbs and 12 right l imbs were involved. The median duration of disease was 2 years (from 3 months to9 years). Intermittent claudication was observed in 5 cases (5 l imbs), 16 patients (17 l imbs) had symptom of rest pain, 4 patients (5 l imbs) suffered ulcer on the distal l imbs. The results of visual analogue scale (VAS), maximum walking distance (MWD), ankle/brachial index (ABI), and transcutaneous oxygen pressure (TcPO2) before operation were (7.16 ± 1.12) points, (0.098 ± 0.043) km, 0.20 ± 0.09, and (11.78 ± 3.46) mm Hg (1 mm Hg=0.133 kPa), respectively. A total of 300 mL bone-marrow blood was extracted from the il iac bone. And then the mononuclear cells were isolated from the bone-marrow blood. All patients received cell transplantation only one time. The amount of transplantation bone marrow mononuclear cells was (1.82-29.46) × 109 (mean 13.33 × 109). Results All patients were followed up for 1 years. After 4 weeks of implantation, the results of VAS, MWD, ABI, and TcPO2 were (2.39 ± 0.51) points, (0.783 ± 0.176) km, 0.28 ± 0.16, (21.33 ± 6.57) mm Hg, respectively, showing significant difference compared with preoperative results (P lt; 0.05). The VAS, MWD, ABI, and TcPO2 increased to (2.44 ± 0.67) points, (1.199 ± 0.304) km, 0.37 ± 0.09, (27.90 ± 5.23) mm Hg after 1 year of implantation, showing significant differences compared with preoperative results (P lt; 0.05). One ulcer healed well and the improvement was obtained in other 3 cases after 4 weeks of implantation (80%). Four ulcers healed well after 1 year of implantation (80%). After 1 year of implantation, angiography revealed 37.04% affected limbs had a satisfactory neovascularization. The angiographic levels were grade 0 in 5 cases, grade 1 in 12 cases, grade 2 in 4 cases, and grade 3 in 6 cases. Conclusion Autologous bone marrow mononuclear cells transplantation could be a simple, safe, effective method to treat TAO.
Objective To explore the middle-term outcome of autologous bone marrow mononuclear cells transplantation in the treatment of lower l imb ischemia. Methods From March 2003 to June 2005, 65 patients with lower l imb ischemia were treated by autologous bone marrow mononuclear cells transplantation. Of the patients, there were 50 males and 15 females, with a mean age of 66.5 years (range 36-89 years), including 4 cases of simple arteriosclerotic occlusion,5 cases of thromboangiitis obl iterans and 56 cases diabetic lower l imb ischemia. A total of 400 mL bone-marrow blood were extracted from the posterior superior il iac crest. And then the mononuclear cells were isolated from the bone-marrow blood in the laboratory. The amount of transplantation bone marrow mononuclear cells was (0.60-1.80) × 109 (mean 1.05 × 109). Twelve patients received cell transplantation from two to four times and the other patients one time. According to the improvement of cl inical finding, the outcome was evaluated. Results All the patients were followed up for 8-56 months (mean 21.5 months). There were 8 deaths, and the mortal ity was 12.3%; 5 were due to myocardial infarction and heart failure and 3 were due to cerebral infarction. The general effective rate was 70.8% (46/65) and the recurrent rate was 10.7% (7/65). Of them, the response to treatment lasted over 12 months in 42 cases, accounting for 91.3% (42/46); over 24 months in 24 cases, accounting for 52.2% (24/46); and over 37 months in 12 cases, accounting for 26.1% (12/46). The effective rates were 100% in 12 patients who received 2-4 times transplantation and 64.2% in 53 patients who received 1 time transplantation, showing statistically significant difference between them (P lt; 0.001). Conclusion The middle-term outcome of autologous bone marrow mononuclear cells transplantation show that it is a feasible and simple method for treatment of lower l imb ischemia.
Objective To observe the clinical efficiency of the implantation of the autologous bone marrow mononuclear cells for treatment of lower limb ischemia after the bone marrow stimulation. Methods From May to December 2005, 43 ischemic limbs in 35 patients (23 males,12 females; aged 3490 years,averaged 71.3 year) were treated. Of the 35 patients, 30 had diabetic lowerlimb ischemia with 38 lower ischemic limbs, 2 had atherosclerosis obliterans with 2 ischemic lower limbs, and 3 had thromboangiitis obliterans with 3 ischemic lower limbs. Five patients with 5 ischemic limbs were in stage Ⅰ lower limb ischemia (intermittentclaudication), 15 patients with ischemic 19 limbs were in stage Ⅱ (rest pain),9 patients with 12 ischemic limbs were in stage Ⅲa(ulceration), and 6 patients with 7 ischemic lower limbs in stage Ⅲb (gangrene); 88.4% of all the ischemic lower limbs (38/43)had a pain, 79.1%(34/43) had coldness, and 69.8%(30/43)had limb numbness. The bone marrow of each patient was stimulated by an injection of the recombinant human granulocyte-macrophage colony-stimulatory factor 300 μg/d for 2-3 days. The bone marrow 130-200 ml was drawn from the iliac spine and the mononuclear cells were obtained. Each patient received implantation of the autologous bone marrow mononuclear cells by an intramuscular injection, an arterial intraluminal injection or a combined injection of the two routes.Results The pain relief was found in 94.7% of theischemic lower limbs, and pain improvement in 97.1% . Relived numbness was found in 93.3%. The distance of the claudication was increased by all the ischemic limbs. An increase in the ankle/ brachial index (ABI)was found in 47.9%. The transcutaneous oxygen pressure (TcPO2) increased in 92.3%. The ulcer heal rate was 9.1% (1/11). Markedlyreduced ulcer wound was found in 27.3% (3/11). The amputation rate was 6.3% (3/48). Arterial angiography revealed that there was a new collateral vessel formationin 91.2%. Complications were as follows: fever and mild fatigue-developed respectively in 1 patient after the bone marrow stimulation, but relieved by themselves. Acute but mild myocardial infarction was found in 1 patient with a slight precordial pain and elevation of myocardial enzymes 1 week after transplantation of the bone marrow mononuclear cells, but recovered after medical treatment. The follow-up averaged 5 months. According to the subjective criteria, the overall efficacy was90%. ABI increased in 62.5% of the patients after operation and the value of TcPO2 was higher in 90% of the patients after this kind of therapy. Arterial angiography revealed a new collateral vessel formation in 90.5% of the 21 ischemic limbs. The foot ulcer healed in 7 and obviously improved in 3. Three of the foot ulcer patients were discharged 2-3 months after the amputation was performed on the diseased toes. Conclusion Implantation of the autologous bone marrow mononuclear cells after the bone marrow stimulation of treatment of the lower limb ischemia has advantages of less marrow aspiration, more mononuclear cell content, satisfactory shortterm effect, and relatively high safety. Itis a new method of treating the lower limb ischemia besides the autologous bone marrow and peripheral blood mononuclear cell implantation. The longterm effect of this method needs a further study.
【摘要】 目的 了解乙型肝炎病毒(HBV)X基因在HBV相关肝病肝移植受体术后外周单个核细胞(PBMC)和骨髓CD34+细胞内的整合情况及其对乙肝疫苗接种的影响。 方法 采集1999年6月-2005年11月25例HBV相关肝病肝移植受体的外周静脉血及其中23例的骨髓血,以密度梯度离心结合单克隆免疫磁珠分离法获取外周血单个核细胞及骨髓CD34+细胞后,提取细胞DNA。因HBV X基因的整合频率最高,设计HBV X基因的特异引物,进行HBV-Alu-PCR,终产物进行电泳并回收、连接载体、筛选扩增后测序,检测有无X基因整合。 结果 经PCR后电泳及测序分析,25例HBV相关肝病肝移植受体术后的PBMC内未检测出HBV X基因的整合,其中采集到骨髓标本的23例CD34+细胞中亦未检测到HBV X基因的整合。 结论 肝移植术后受体体内HBV微生态的剧烈改变,使HBV整合的基本条件丧失,在此情况下,外周免疫细胞及骨髓造血干/祖细胞不是发生HBV整合的适宜场所,乙肝疫苗接种效果与HBV X基因整合关系不明确。【Abstract】 Objective To investigate whether hepatitis B virus HBV X gene integrates in peripheral blood mononuclear cell (PBMC) and bone marrow CD34+ cells from HBV related liver disease recipients after liver transplantation. Methods Between June 1999 and November 2005, PBMC were obtained from 25 HBV related liver disease recipients after liver transplantation and bone marrow CD34+ cells obtained from 23 cases among them. The cellular DNA was extracted by DNA isolation and purification kit following the manufacture’s instructions. Specific primers to HBV X gene and to human Alu repeats were used to amplify the virus integration through a 3-round hemi-nest PCR. The PCR final product was judged by 1.2% agarose electrophoresis, ligated to T vector, proliferated in E. coil 5α and sequenced. Results According to agarose electrophoresis and sequencing analysis, there were no HBV X gene integration in PBMC and bone marrow CD34+ cells from HBV related liver transplant recipients after surgery. Conclusions Because of the radical change of HBV microecological environment in HBV related liver transplant recipients after operation, the fundamental condition of HBV integration has been lost, which led PBMC and bone marrow CD34+ cells not suit to HBV X gene integrate to human genome. And the impact of HBV X gene integration on HBV vaccination is still undefined.
【摘要】 目的 检测B细胞成熟抗原(BCMA)mRNA在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)的表达水平,探讨BCMA在SLE发病中的意义。 方法 纳入2006年1-11月收治的36例SLE患者,同期17例健康志愿者作为对照组,采用半定量RT-PCR法检测外周血单个核细胞中BCMA mRNA的表达,并与SLE疾病活动指数(SLEDAI)进行相关性分析。 结果 SLE患者组BCMA mRNA表达水平(0.598±0.230)均明显高于正常对照组(0.411±0.309)(Plt;0.05)。SLE患者BCMA mRNA表达水平与SLEDAI评分无相关性(P=0.590)。 结论 SLE患者BCMA mRNA表达水平的增高,可能在SLE的发病机制中具有一定的作用。【Abstract】 Objective To detect the mRNA expression of B-cell maturation antigen (BCMA) in peripheral blood mononuclear cells (PBMC) in patients with systemic lupus erythematosus (SLE), and explore the role of BCMA in the pathogenesis of SLE. Methods From January 2006 to November 2006 the expression of BCMA mRNA in PBMC of 36 patients with SLE and 17 normal controls were measured by half-quantitative RT-PCR. The linear correlation between the expression of BCMA mRNA and SLE disease activity index (SLEDAI) was assessed. Results The level of BCMA mRNA (0.598±0.230) in PBMC significantly increased in SLE patients compared with that in the normal controls (0.411±0.309) (Plt;0.05). The expression of BCMA mRNA in SLE patients showed no correlation with SLEDAI score (P=0.590). Conclusion The results suggest that the expression of BCMA mRNA might play an important role in the pathogenesis of SLE.