CA诱导早熟凝集染色体作为生物剂量计的研究

发布时间：2018-05-15 20:15

本文选题：染色体畸变 + 早熟凝集染色体　；参考：《济南大学》2013年硕士论文

【摘要】：目的 1探索用calyculinA诱导外周血淋巴细胞发生早熟染色体凝集的最优化方案。 2探讨CalyculinA诱导早熟染色体凝集技术作为低剂量电离辐射生物剂量计的可行性。 3为了解决低传能线密度辐射诱发的染色体畸变存在剂量率效应的问题,分别观察不同剂量率条件下的剂量效应关系,并建立相应的剂量效应曲线和数学模型,以便较为准确地估算不同剂量率受照人员的生物剂量。 4通过运用CalyculinA诱导PCC技术,探索QPCC法估算6MV X射线局部受照份额与剂量的可行性。方法 1抽取健康成年献血员外周血6ml,分别接种于12瓶预先配置好的RPMI-1640培养基(20%的小牛血清、1%的PHA,1%的HEPES)中,然后随机分成3’组并编号。培养48h后收获制片,培养至24h时加入秋水仙素,继续培养至46h时加入CalyculinA。采用析因实验设计对影响早熟凝集染色体形态和数量的两个主要因素秋水仙素和CalyculinA分别进行两个水平上的探讨,并应用相关统计学方法对分裂指数(MI)进行分析,以此判断最优方案。 2抽取健康成年人的抗凝外周静脉血,用X射线分别照射0、0.1Gy、0.25Gy、0.5Gy、0.75Gy和1Gy。剂量率为400MU/min照射后将血样接种于RPMI1640培养基中于37℃、5%C02恒温培养箱内培养48h,当培养24h时加入秋水仙素,终止培养前2h加入CalyculinA诱导早熟凝集染色体,收获制片并用Giemsa染液染色,观察G_1、G_2/M-PCC细胞中总畸变率、断片率、双着丝粒体加着丝粒环率与辐射剂量的效应关系。采用盲法阅片,每个剂量点分析1000个G_1、G_2/M-PCC细胞,计数各剂量组染色体畸变数,应用SPSS16.0统计软件包进行统计分析。采用WHO提供的3种数学模式对实验数据做曲线拟合,通过检验回归系数的显著性和曲线的拟合度确定拟合曲线。 3抽取健康成年人的抗凝外周静脉血6m1,肝素抗凝后分装于12个0.5m1的EP管中密封,分成2组,每组6个,将EP管放入固体水的插孔中,剂量率分别采用100Mu/min和200Mu/min。用X射线分别照射0、0.1Gy、0.25Gy、0.5Gy、0.75Gy和1Gy。然后接种于RPMI1640培养基中,放置于37℃恒温箱内培养48h,当培养24h时加入秋水仙素,终止培养前2h加入CalyculinA诱导早熟凝集染色体,收获制片并用Giemsa染液染色,采用盲法阅片,计数各标本的染色体双着丝粒+环畸变率。应用SPSS16.0统计软件包分析染色体畸变率与照射剂量之间的关系,分别拟合不同剂量率的剂量效应曲线和数学模型。 4抽取健康成年人的抗凝外周静脉血,用X射线分别照射0、5Gy、10Gy、15Gy、20Gy和25Gy。,培养52h并用CalyculinA诱导PCC。记录各剂量点PCC指数、PCC环以及断片数,判断观察到的PCC环是否符合泊松分布,分别建立相应的PCC环率和断片率的剂量效应关系曲线。同时用8Gy6MV X射线照射离体健康人外周血,受照份额为20%、50%、80%模拟局部照射。结果 1不同水平的秋水仙素和CalyculinA培养的标本：淋巴细胞活化佳,CalyculinA终浓度高的的实验组获得的分裂指数明显高于CalyculinA终浓度低的实验组,CalyculinA终浓度的不同水平影响的差异具有统计学意义(P0.01),秋水仙素终浓度的不同水平影响的差异同样具有统计学意义(P0.05)。 2用CalyculinA可成功诱导人外周血淋巴细胞产生PCC,且总畸变率、断片率、双着丝粒体加着丝粒环率随照射剂量的增加而增加,呈现出明显的剂量效应关系。根据不同剂量X射线照射后诱发的PCC畸变率统计结果,用SPSS16.0统计软件拟合其剂量效应的回归方程,计算方程拟合度(R2),检验回归系数的显著性。结果表明,在0～1.OGy剂量范围内,断片率、双着丝粒体+着丝粒环率、总畸变率拟合的最佳数学模式,均为二次多项式：y＝a＋bD＋cD2。 3在剂量率一定的情况下,各剂量点的(dic+r)率随吸收剂量增加而增加。同一剂量点不同剂量率射线诱发的(dic+r)畸变率随剂量率的增加而增加,剂量率效应明显。 4统计结果发现PCC指数随着剂量水平的增高而降低,各剂量点样品中PCC环分布符合泊松分布。PCC环率与断片率都随着受照剂量增加而增加。每个受损细胞所含多余PCC断片数与照射剂量之间符合线性模式,其数值可被用于模拟局部照射的剂量估算。结论 1淋巴细胞培养24h之后加秋水仙素使其终浓度达0.04μg/ml,继续培养至48h收获,与收获前2h加calyculinA使其终末浓度达60nM,可以获得较高比例的分裂指数,容易计数,可对原始损伤作出较准确的估计。 2早熟染色体凝集技术可以用于估算低剂量受照者辐射剂量。 3在估算低传能线密度辐射的吸收剂量时,必须考虑剂量率效应,应根据辐照情况尽量选择剂量率相近的剂量-效应关系曲线进行估算,从而使结果更为准确。 4采用QPCC的方法可以较准确地估算活体局部受照剂量与份额,可用于局部大剂量电离损伤程度测定。
[Abstract]:objective
1 explore the optimal scheme of inducing premature chromosome condensation with peripheral blood lymphocytes induced by calyculinA.
2 to explore the feasibility of CalyculinA induced premature chromosome agglutination as a low dose ionizing radiation biodosimeter.
3 in order to solve the problem of the dose rate effect of chromosome aberration induced by low energy line density radiation, the dose effect relationship under different dose rates was observed respectively, and the corresponding dose effect curve and mathematical model were established in order to accurately estimate the biological dose of the persons exposed to different dose rates.
4 by using CalyculinA to induce PCC technology, we explored the feasibility of QPCC method to estimate the share and dose of 6MV X ray local exposure.
Method
1 the peripheral blood 6ml of healthy adult blood donors was selected and inoculated into 12 bottles of pre configured RPMI-1640 medium (20% calf serum, 1% PHA, 1% HEPES) and then randomly divided into 3 'group and numbered. After cultivating 48h, the production was harvested and colchicine was added to 24h, and the factorial design was adopted to add CalyculinA. to 46h after continuous culture, and the factorial design was adopted. The two main factors, colchicine and CalyculinA, which affect the morphology and quantity of precocious agglutination chromosomes, were discussed at two levels, and the correlation statistical method was used to analyze the split index (MI), so as to determine the optimal scheme.
2 the blood of anticoagulant peripheral vein of healthy adults was irradiated with X ray, 0,0.1Gy, 0.25Gy, 0.5Gy, 0.75Gy and 1Gy. were irradiated by 400MU/min, and the blood samples were inoculated in RPMI1640 medium at 37, and 48h in the incubator of 5%C02 constant temperature. When the incubator was incubated, the daffodil was added to the incubator, and the precocious agglutination was induced before the culture was terminated. Chromosomes were harvested and stained with Giemsa dye to observe the total aberration rate, fragment rate of G_1 and G_2/M-PCC cells, the relationship between the ratio of double centromere plus centromere ring and radiation dose. Using blind method, 1000 G_1, G_2/M-PCC cells were analyzed at each dose point, and the number of chromosomal aberrations in each dose group was counted, and the SPSS16.0 statistical software package was used. The 3 mathematical models provided by WHO are used to curve the experimental data, and the fitting curves are determined by testing the significance of the regression coefficient and the fitting degree of the curves.
3 the anticoagulant peripheral venous blood 6m1 was extracted from healthy adults, and heparin was sealed in 12 0.5m1 EP tubes after anticoagulation, divided into 2 groups, 6 in each group. The EP tube was placed in the socket of solid water. The dose rate was respectively irradiated with 100Mu/min and 200Mu/min. with X rays, respectively, and 0.25Gy, 0.5Gy, 0.75Gy and 1Gy. were then inoculated in the medium. At 37 centigrade incubator, 48h was cultured, colchicine was added when 24h was cultured, 2h was added to CalyculinA to induce precocious agglutination chromosomes before culture, and CalyculinA was stained with Giemsa dye solution. The chromosome aberration rate and chromosome aberration rate of chromosomes were counted by blind method. The chromosome aberration rate and irradiation were analyzed by SPSS16.0 software package. The dose effect curves and mathematical models of different dose rates were fitted respectively.
4 the blood of anticoagulant peripheral vein of healthy adults was taken. 0,5Gy, 10Gy, 15Gy, 20Gy and 25Gy. were irradiated with X ray respectively. The 52h was cultured and the PCC index of each dose point, PCC ring and the number of fragments were recorded with CalyculinA, to determine whether the observed PCC ring conforms to the Poisson distribution, and the corresponding dose effect relationship of the ring rate and the fragment rate was established. The irradiated peripheral blood of healthy individuals was irradiated with 8Gy6MV X rays at the same time. The irradiated share was 20%, 50%, 80% simulated local irradiation.
Result
1 different levels of colchicine and CalyculinA culture specimens: the lymphocyte activation is good, the CalyculinA terminal concentration is higher than that of the experimental group with low end concentration of CalyculinA, and the difference of the different levels of the CalyculinA terminal concentration is statistically significant (P0.01), and the final concentration of colchicine is different water. The difference of flat effects was also statistically significant (P0.05).
2 CalyculinA can successfully induce human peripheral blood lymphocytes to produce PCC, and the total aberration rate, fragment rate and the rate of double centromere plus centromere ring increase with the increase of irradiation dose, showing a significant dose effect relationship. According to the statistical results of PCC distortion induced by different doses of X ray irradiation, the dosage of SPSS16.0 statistical software is used to fit the dose. The regression equation of the effect, the fitting degree of the equation (R2) was calculated and the significance of the regression coefficient was tested. The results showed that the best mathematical models of the fragment rate, the double centromere + centromere ring rate and the total aberration rate in the range of 0 ~ 1.OGy were all two times polynomial: y = a + bD + CDC.
3 at a certain dose rate, the (dic+r) rate of every dose point increases with the increase of the absorbed dose. The rate of radiation induced (dic+r) distortion at the same dose rate increases with the increase of dose rate, and the dose rate effect is obvious.
4 the statistical results show that the PCC index decreases with the increase of the dose level. The PCC ring distribution in each dose point sample increases with the increasing of the Poisson distribution.PCC ring rate and the fragment rate. The number of superfluous PCC fragments in each damaged cell is in line with the irradiated dose, and its value can be used to simulate local irradiation. Dose estimation.
conclusion
After 1 lymphocyte culture, colchicine was added with colchicine to make its final concentration of 0.04 Mu and continue to be cultured to 48h harvest, and 2H plus calyculinA before harvest made its end concentration 60nM, which could obtain a high proportion of split index, which was easy to count, and could make a more accurate estimation of the original damage.
2 the premature chromosome agglutination technique can be used to estimate the radiation dose of low dose recipients.
3 when estimating the absorbed dose of low energy line density radiation, the dose rate effect must be taken into consideration. The dose effect relationship curve of similar dose rate should be chosen according to the irradiation condition as far as possible, so that the result is more accurate.
4 the QPCC method can be used to estimate the dose and fraction of local exposure more accurately, and it can be used for the determination of local large dose ionization injury.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R144.1

【参考文献】