本文選題：薄芝糖肽 + 胃癌��； 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：目的:1.探討密度梯度離心法分離胃癌患者外周血單個核細胞(Peripheral blood mononuclear cells,PBMCs)的優(yōu)化條件,為細胞分離提供質(zhì)量保證。2.利用數(shù)學模型,分析薄芝糖肽濃度、細胞培養(yǎng)時間、是否為胃癌患者的對象類別共3個因素對PBMCs增殖的影響,初步明確薄芝糖肽調(diào)節(jié)胃癌患者PBMCs增殖的可能性。3.探索薄芝糖肽對胃癌患者PBMCs增殖調(diào)節(jié)的規(guī)律。4.明確薄芝糖肽對胃癌患者PBMCs分泌抑制性細胞因子白細胞介素-10(Interleukin-10,IL-10)的影響。方法:1.用JMP 10.0軟件的定制實驗設(shè)計模塊,以離心時間、離心速度為因子變量,離心所得PBMCs的厚度值為響應變量,定義因子約束,添加因子的交互效應及二次效應,按照定制設(shè)計的離心時間和速度組合條件,使用Filcoll淋巴細胞分離液分離PBMCs,數(shù)碼相機拍攝離心管中的細胞分層,Photoshop CS 6軟件測量PBMCs層的厚度,JMP軟件最小二乘法分析、最大化意愿因子刻畫獲得最佳離心條件,驗證離心,測量PBMCs厚度值,臺盼藍染色,計算細胞存活率。2.用JMP 10.0軟件定制設(shè)計方法,以藥物濃度(mg/L),培養(yǎng)時間(h),對象類別(研究對象的分類水平為:胃癌患者、非胃癌患者)作為因子變量,PBMCs的吸光度值(OD)作為響應變量,對藥物濃度、培養(yǎng)時間2個連續(xù)變量進行因子約束,根據(jù)定制設(shè)計條件進行MTT檢測,測定不同條件組合模式對應的PBMCs的OD值,建立數(shù)學模型,標準最小二乘法進行效應篩選,進行擬合模型分析。3.采用重復測量數(shù)據(jù)的分差分析方法,根據(jù)薄芝糖肽濃度分為6組(0 mg/L即對照組,25 mg/L、50 mg/L、100 mg/L、150 mg/L、200 mg/L藥物干預組),分別作用于胃癌患者的PBMCs,分別培養(yǎng)4小時、12小時、24小時、48小時,MTT法測定OD值,JMP 10.0軟件分析結(jié)果。4.分4組干預胃癌患者PBMCs(16小時),薄芝糖肽的每組終濃度分別為0 mg/L、50 mg/L、100 mg/L、200 mg/L,ELISA法測定IL-10含量。結(jié)果:1.PBMCs離心條件優(yōu)化模型的F值為8.911,P0.01,決定系數(shù)(R2)為0.88。離心速度與離心時間的交互作用對PBMCs厚度的影響無統(tǒng)計學意義(P0.05),離心時間及其二次效應、離心速度及其二次效應對PBMCs厚度的影響均具有統(tǒng)計學意義(P0.05)。擬合模型因子刻畫的曲面圖顯示:離心時間、離心速度與離心厚度呈現(xiàn)較為復雜的曲線關(guān)系,而非簡單的線性關(guān)聯(lián)。最優(yōu)離心條件為:離心時間=10 min,離心速度=2307 r/min。優(yōu)化條件下分離PBMCs,臺盼藍染色,計算細胞存活率平均為95%。2.薄芝糖肽濃度與細胞培養(yǎng)時間的交互作用對PBMCs的增殖影響具有統(tǒng)計學意義,P0.05。藥物濃度對增殖的影響具有統(tǒng)計學意義,P0.01。培養(yǎng)時間主效應的P=0.055,對象類別的主效應、與時間和濃度的交互效應均無統(tǒng)計學意義(P0.05)。等高線圖顯示時間因素的作用較小。因子軌跡線顯示濃度因子的陡峭程度明顯高于培養(yǎng)時間、對象類別。最大化意愿刻畫的薄芝糖肽濃度為196 mg/L、培養(yǎng)時間為4 h、對象類別為胃癌患者,OD值為0.354308,95%CI(0.30289,0.40573)。3.重復測量數(shù)據(jù)方差分析模型的Mauchly準則為0.781,P0.01;時間、濃度的交互作用校正P值0.01。對象內(nèi)效應(時間)的F值為286.077,P值為2.553E-43,一元的G-G Epsilon、一元的H-F Epsilon校正檢驗顯示,對象內(nèi)效應的差異具有統(tǒng)計學意義(P0.01)。對象間效應(濃度)的F值為192.390,P值為1.894E-44。4個時間點內(nèi)部,與對照組比較,不同濃度的薄芝糖肽均可促進胃癌患者PBMCs的增殖(P0.05)。4.0 mg/L、50 mg/L、100 mg/L、200 mg/L組的IL-10濃度(`x±s,pg/ml)分別為:(5.36±0.46)、(6.73±0.25)、(9.23±0.77)、(12.35±0.86)。單因素方差分析得出:50 mg/L組與對照組對比,P0.05;100 mg/L、200 mg/L組分別與對照組對比,P0.01;100 mg/L組、200 mg/L組分別與50 mg/L組比較,P0.01;200 mg/L組與100 mg/L組比較,P0.01。結(jié)論:1.JMP定制實驗設(shè)計方案可以優(yōu)化胃癌患者PBMCs的離心分離條件,數(shù)學模型分析結(jié)果可以進一步進行最大化意愿刻畫,獲得最佳的分離參數(shù)值,驗證離心顯示PBMCs存活率較高。2.薄芝糖肽濃度、培養(yǎng)時間、對象類別(胃癌和非胃癌患者)的多因子模型中,濃度對PBMCs的增殖發(fā)揮影響。胃癌患者的PBMCs對薄芝糖肽的促增殖反應與非胃癌患者無顯著性差異,存在使用薄芝糖肽調(diào)節(jié)胃癌患者PBMCs增殖的基礎(chǔ)。3.薄芝糖肽濃度依賴性和時間依賴性地促進胃癌患者PBMCs的增殖。4.薄芝糖肽促進胃癌患者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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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