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薄芝糖肽对胃癌患者PBMCs增殖及IL-10分泌的作用

发布时间:2018-06-28 07:07

  本文选题:薄芝糖肽 + 胃癌 ; 参考:《安徽医科大学》2017年硕士论文


【摘要】:目的:1.探讨密度梯度离心法分离胃癌患者外周血单个核细胞(Peripheral blood mononuclear cells,PBMCs)的优化条件,为细胞分离提供质量保证。2.利用数学模型,分析薄芝糖肽浓度、细胞培养时间、是否为胃癌患者的对象类别共3个因素对PBMCs增殖的影响,初步明确薄芝糖肽调节胃癌患者PBMCs增殖的可能性。3.探索薄芝糖肽对胃癌患者PBMCs增殖调节的规律。4.明确薄芝糖肽对胃癌患者PBMCs分泌抑制性细胞因子白细胞介素-10(Interleukin-10,IL-10)的影响。方法:1.用JMP 10.0软件的定制实验设计模块,以离心时间、离心速度为因子变量,离心所得PBMCs的厚度值为响应变量,定义因子约束,添加因子的交互效应及二次效应,按照定制设计的离心时间和速度组合条件,使用Filcoll淋巴细胞分离液分离PBMCs,数码相机拍摄离心管中的细胞分层,Photoshop CS 6软件测量PBMCs层的厚度,JMP软件最小二乘法分析、最大化意愿因子刻画获得最佳离心条件,验证离心,测量PBMCs厚度值,台盼蓝染色,计算细胞存活率。2.用JMP 10.0软件定制设计方法,以药物浓度(mg/L),培养时间(h),对象类别(研究对象的分类水平为:胃癌患者、非胃癌患者)作为因子变量,PBMCs的吸光度值(OD)作为响应变量,对药物浓度、培养时间2个连续变量进行因子约束,根据定制设计条件进行MTT检测,测定不同条件组合模式对应的PBMCs的OD值,建立数学模型,标准最小二乘法进行效应筛选,进行拟合模型分析。3.采用重复测量数据的分差分析方法,根据薄芝糖肽浓度分为6组(0 mg/L即对照组,25 mg/L、50 mg/L、100 mg/L、150 mg/L、200 mg/L药物干预组),分别作用于胃癌患者的PBMCs,分别培养4小时、12小时、24小时、48小时,MTT法测定OD值,JMP 10.0软件分析结果。4.分4组干预胃癌患者PBMCs(16小时),薄芝糖肽的每组终浓度分别为0 mg/L、50 mg/L、100 mg/L、200 mg/L,ELISA法测定IL-10含量。结果:1.PBMCs离心条件优化模型的F值为8.911,P0.01,决定系数(R2)为0.88。离心速度与离心时间的交互作用对PBMCs厚度的影响无统计学意义(P0.05),离心时间及其二次效应、离心速度及其二次效应对PBMCs厚度的影响均具有统计学意义(P0.05)。拟合模型因子刻画的曲面图显示:离心时间、离心速度与离心厚度呈现较为复杂的曲线关系,而非简单的线性关联。最优离心条件为:离心时间=10 min,离心速度=2307 r/min。优化条件下分离PBMCs,台盼蓝染色,计算细胞存活率平均为95%。2.薄芝糖肽浓度与细胞培养时间的交互作用对PBMCs的增殖影响具有统计学意义,P0.05。药物浓度对增殖的影响具有统计学意义,P0.01。培养时间主效应的P=0.055,对象类别的主效应、与时间和浓度的交互效应均无统计学意义(P0.05)。等高线图显示时间因素的作用较小。因子轨迹线显示浓度因子的陡峭程度明显高于培养时间、对象类别。最大化意愿刻画的薄芝糖肽浓度为196 mg/L、培养时间为4 h、对象类别为胃癌患者,OD值为0.354308,95%CI(0.30289,0.40573)。3.重复测量数据方差分析模型的Mauchly准则为0.781,P0.01;时间、浓度的交互作用校正P值0.01。对象内效应(时间)的F值为286.077,P值为2.553E-43,一元的G-G Epsilon、一元的H-F Epsilon校正检验显示,对象内效应的差异具有统计学意义(P0.01)。对象间效应(浓度)的F值为192.390,P值为1.894E-44。4个时间点内部,与对照组比较,不同浓度的薄芝糖肽均可促进胃癌患者PBMCs的增殖(P0.05)。4.0 mg/L、50 mg/L、100 mg/L、200 mg/L组的IL-10浓度(`x±s,pg/ml)分别为:(5.36±0.46)、(6.73±0.25)、(9.23±0.77)、(12.35±0.86)。单因素方差分析得出:50 mg/L组与对照组对比,P0.05;100 mg/L、200 mg/L组分别与对照组对比,P0.01;100 mg/L组、200 mg/L组分别与50 mg/L组比较,P0.01;200 mg/L组与100 mg/L组比较,P0.01。结论:1.JMP定制实验设计方案可以优化胃癌患者PBMCs的离心分离条件,数学模型分析结果可以进一步进行最大化意愿刻画,获得最佳的分离参数值,验证离心显示PBMCs存活率较高。2.薄芝糖肽浓度、培养时间、对象类别(胃癌和非胃癌患者)的多因子模型中,浓度对PBMCs的增殖发挥影响。胃癌患者的PBMCs对薄芝糖肽的促增殖反应与非胃癌患者无显著性差异,存在使用薄芝糖肽调节胃癌患者PBMCs增殖的基础。3.薄芝糖肽浓度依赖性和时间依赖性地促进胃癌患者PBMCs的增殖。4.薄芝糖肽促进胃癌患者PBMCs分泌IL-10。
[Abstract]:Objective: 1. to investigate the optimum conditions for the separation of Peripheral blood mononuclear cells (PBMCs) from peripheral blood of gastric cancer patients with density gradient centrifugation, and to provide quality assurance.2. using mathematical model for cell separation, and to analyze the concentration of thin Ganoderma peptide and cell culture time, and whether there are 3 factors for the proliferation of PBMCs in patients with gastric cancer. The effect of thin Ganoderma peptide on the proliferation of PBMCs in gastric cancer patients.3. explore the regulation of PBMCs proliferation in gastric cancer patients.4. clearly the effect of thin Ganoderma on the PBMCs secretion of interleukin-x -10 (Interleukin-10, IL-10) secreted by PBMCs in gastric cancer patients. Method: 1. custom experiment with JMP 10 software Considering the centrifugal time and the centrifugal velocity as the factor variable, the thickness of the centrifuge PBMCs is the response variable, the factor constraint is defined, the interaction effect and the two effect of adding factors are defined, the Filcoll lymphocyte separation solution is used to separate PBMCs, and the digital camera is used to shoot the centrifuge tube. Cell layer, Photoshop CS 6 software measure the thickness of PBMCs layer, JMP software least square analysis, maximum will factor depict the best centrifuge conditions, verify centrifugation, measure PBMCs thickness value, trypan blue staining, calculate cell survival rate.2. using JMP 10 software custom design method, drug concentration (mg/L), culture time (H), object category ( The classification level of the subjects is: gastric cancer patients, non gastric cancer patients as factor variables, the absorbance value (OD) of PBMCs as response variable, factor constraint on 2 continuous variables of drug concentration and culture time, MTT detection according to custom design conditions, and the determination of PBMCs values corresponding to different conditional combination patterns, and a mathematical model is established. The standard least square method was used to screen the effect, and the fitting model was used to analyze the difference analysis method of.3. with repeated measurement data. According to the concentration of thin Ganoderma peptide, it was divided into 6 groups (0 mg/L, the control group, 25 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L drug intervention group), respectively, for 4 hours, 12 hours, 24 hours respectively for the gastric cancer patients. 48 hours, MTT method was used to determine OD value, and JMP 10 software analysis results.4. were divided into 4 groups to intervene PBMCs (16 hours) for gastric cancer patients. The final concentrations of each group were 0 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, ELISA method to determine IL-10 content. The effect of cardiac time interaction on the thickness of PBMCs was not statistically significant (P0.05). The effect of centrifugal time and its two effects, centrifugal velocity and its two effects on PBMCs thickness were all statistically significant (P0.05). The curved surface diagram depicted by the fitting model factor showed that the centrifugal velocity and the centrifugal thickness showed a more complex curve during centrifugal time. The optimal centrifugal condition is: centrifugation time =10 min, centrifuge speed =2307 r/min. optimization, PBMCs separation, trypan blue staining, and the mean cell survival rate is 95%.2. thin Ganoderma concentration and cell culture time interaction effect on PBMCs proliferation is statistically significant, P0.05. drug concentration The effect on proliferation has statistical significance, the main effect of P0.01. culture time, the main effect of P=0.055, the main effect of the object category, and the interaction effect of time and concentration have no statistical significance (P0.05). The contour map shows that the effect of time factor is smaller. The factor locus line shows that the steepness of the concentration factor is obviously higher than that of the culture time and the object category. The concentration of the glycopeptide was 196 mg/L, the incubation time was 4 h, the target category was gastric cancer patients, and the Mauchly criterion of 0.354308,95%CI (0.30289,0.40573).3. repeated measurement data variance analysis model was 0.781, P0.01; time and the interaction of concentration corrected the F value of the P value internal effect (time) of 286.077 and P. For 2.553E-43, one yuan G-G Epsilon, one yuan H-F Epsilon correction test showed that the difference in the effect in the object was statistically significant (P0.01). The F value of the inter object effect (concentration) was 192.390 and the P value was within the 1.894E-44.4 time point. Compared with the control group, the different concentration of thin Ganoderma peptide could promote the proliferation of PBMCs in gastric cancer patients (P0.05).4.0. Mg/L, 50 mg/L, 100 mg/L, and 200 mg/L groups (`x + s, pg/ml) were respectively (5.36 + 0.46), (6.73 + 0.25), (9.23 + 0.77), (12.35 + 0.86). Single factor analysis of variance analysis showed that the 50 mg/L group was compared with the control group, P0.05; 100 mg/L, 200 mg/L groups compared with the control group Group /L and 100 mg/L group, P0.01. conclusion: 1.JMP customized experimental design scheme can optimize the centrifugation conditions of PBMCs in gastric cancer patients. The result of mathematical model analysis can further maximize the will depiction, obtain the best separation parameter value, verify the concentration of.2. thin Ganoderma peptide, culture time and object class of PBMCs survival rate of centrifugation. Concentration affects the proliferation of PBMCs in the multifactor model of patients with gastric cancer and non gastric cancer. The proliferation of PBMCs in gastric cancer patients has no significant difference with those of non gastric cancer patients. There is a dependence and time dependence of.3. thin Ganoderma concentration on the proliferation of PBMCs in gastric cancer patients by using thin ganoderma peptide to promote gastric cancer. Proliferation of PBMCs in cancer patients.4. thin glycopeptide promotes PBMCs secretion in gastric cancer patients IL-10.
【学位授予单位】:安徽医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R735.2

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