本文選題：染色體畸變 + 早熟凝集染色體��； 參考：《濟(jì)南大學(xué)》2013年碩士論文

【摘要】：目的 1探索用calyculinA誘導(dǎo)外周血淋巴細(xì)胞發(fā)生早熟染色體凝集的最優(yōu)化方案。 2探討CalyculinA誘導(dǎo)早熟染色體凝集技術(shù)作為低劑量電離輻射生物劑量計的可行性。 3為了解決低傳能線密度輻射誘發(fā)的染色體畸變存在劑量率效應(yīng)的問題,分別觀察不同劑量率條件下的劑量效應(yīng)關(guān)系,并建立相應(yīng)的劑量效應(yīng)曲線和數(shù)學(xué)模型,以便較為準(zhǔn)確地估算不同劑量率受照人員的生物劑量。 4通過運(yùn)用CalyculinA誘導(dǎo)PCC技術(shù),探索QPCC法估算6MV X射線局部受照份額與劑量的可行性。 方法 1抽取健康成年獻(xiàn)血員外周血6ml,分別接種于12瓶預(yù)先配置好的RPMI-1640培養(yǎng)基(20%的小牛血清、1%的PHA,1%的HEPES)中,然后隨機(jī)分成3’組并編號。培養(yǎng)48h后收獲制片,培養(yǎng)至24h時加入秋水仙素,繼續(xù)培養(yǎng)至46h時加入CalyculinA。采用析因?qū)嶒?yàn)設(shè)計對影響早熟凝集染色體形態(tài)和數(shù)量的兩個主要因素秋水仙素和CalyculinA分別進(jìn)行兩個水平上的探討,并應(yīng)用相關(guān)統(tǒng)計學(xué)方法對分裂指數(shù)(MI)進(jìn)行分析,以此判斷最優(yōu)方案。 2抽取健康成年人的抗凝外周靜脈血,用X射線分別照射0、0.1Gy、0.25Gy、0.5Gy、0.75Gy和1Gy。劑量率為400MU/min照射后將血樣接種于RPMI1640培養(yǎng)基中于37℃、5%C02恒溫培養(yǎng)箱內(nèi)培養(yǎng)48h,當(dāng)培養(yǎng)24h時加入秋水仙素,終止培養(yǎng)前2h加入CalyculinA誘導(dǎo)早熟凝集染色體,收獲制片并用Giemsa染液染色,觀察G_1、G_2/M-PCC細(xì)胞中總畸變率、斷片率、雙著絲粒體加著絲粒環(huán)率與輻射劑量的效應(yīng)關(guān)系。采用盲法閱片,每個劑量點(diǎn)分析1000個G_1、G_2/M-PCC細(xì)胞,計數(shù)各劑量組染色體畸變數(shù),應(yīng)用SPSS16.0統(tǒng)計軟件包進(jìn)行統(tǒng)計分析。采用WHO提供的3種數(shù)學(xué)模式對實(shí)驗(yàn)數(shù)據(jù)做曲線擬合,通過檢驗(yàn)回歸系數(shù)的顯著性和曲線的擬合度確定擬合曲線。 3抽取健康成年人的抗凝外周靜脈血6m1,肝素抗凝后分裝于12個0.5m1的EP管中密封,分成2組,每組6個,將EP管放入固體水的插孔中,劑量率分別采用100Mu/min和200Mu/min。用X射線分別照射0、0.1Gy、0.25Gy、0.5Gy、0.75Gy和1Gy。然后接種于RPMI1640培養(yǎng)基中,放置于37℃恒溫箱內(nèi)培養(yǎng)48h,當(dāng)培養(yǎng)24h時加入秋水仙素,終止培養(yǎng)前2h加入CalyculinA誘導(dǎo)早熟凝集染色體,收獲制片并用Giemsa染液染色,采用盲法閱片,計數(shù)各標(biāo)本的染色體雙著絲粒+環(huán)畸變率。應(yīng)用SPSS16.0統(tǒng)計軟件包分析染色體畸變率與照射劑量之間的關(guān)系,分別擬合不同劑量率的劑量效應(yīng)曲線和數(shù)學(xué)模型。 4抽取健康成年人的抗凝外周靜脈血,用X射線分別照射0、5Gy、10Gy、15Gy、20Gy和25Gy。,培養(yǎng)52h并用CalyculinA誘導(dǎo)PCC。記錄各劑量點(diǎn)PCC指數(shù)、PCC環(huán)以及斷片數(shù),判斷觀察到的PCC環(huán)是否符合泊松分布,分別建立相應(yīng)的PCC環(huán)率和斷片率的劑量效應(yīng)關(guān)系曲線。同時用8Gy6MV X射線照射離體健康人外周血,受照份額為20%、50%、80%模擬局部照射。 結(jié)果 1不同水平的秋水仙素和CalyculinA培養(yǎng)的標(biāo)本：淋巴細(xì)胞活化佳,CalyculinA終濃度高的的實(shí)驗(yàn)組獲得的分裂指數(shù)明顯高于CalyculinA終濃度低的實(shí)驗(yàn)組,CalyculinA終濃度的不同水平影響的差異具有統(tǒng)計學(xué)意義(P0.01),秋水仙素終濃度的不同水平影響的差異同樣具有統(tǒng)計學(xué)意義(P0.05)。 2用CalyculinA可成功誘導(dǎo)人外周血淋巴細(xì)胞產(chǎn)生PCC,且總畸變率、斷片率、雙著絲粒體加著絲粒環(huán)率隨照射劑量的增加而增加,呈現(xiàn)出明顯的劑量效應(yīng)關(guān)系。根據(jù)不同劑量X射線照射后誘發(fā)的PCC畸變率統(tǒng)計結(jié)果,用SPSS16.0統(tǒng)計軟件擬合其劑量效應(yīng)的回歸方程,計算方程擬合度(R2),檢驗(yàn)回歸系數(shù)的顯著性。結(jié)果表明,在0～1.OGy劑量范圍內(nèi),斷片率、雙著絲粒體+著絲粒環(huán)率、總畸變率擬合的最佳數(shù)學(xué)模式,均為二次多項(xiàng)式：y＝a＋bD＋cD2。 3在劑量率一定的情況下,各劑量點(diǎn)的(dic+r)率隨吸收劑量增加而增加。同一劑量點(diǎn)不同劑量率射線誘發(fā)的(dic+r)畸變率隨劑量率的增加而增加,劑量率效應(yīng)明顯。 4統(tǒng)計結(jié)果發(fā)現(xiàn)PCC指數(shù)隨著劑量水平的增高而降低,各劑量點(diǎn)樣品中PCC環(huán)分布符合泊松分布。PCC環(huán)率與斷片率都隨著受照劑量增加而增加。每個受損細(xì)胞所含多余PCC斷片數(shù)與照射劑量之間符合線性模式,其數(shù)值可被用于模擬局部照射的劑量估算。 結(jié)論 1淋巴細(xì)胞培養(yǎng)24h之后加秋水仙素使其終濃度達(dá)0.04μg/ml,繼續(xù)培養(yǎng)至48h收獲,與收獲前2h加calyculinA使其終末濃度達(dá)60nM,可以獲得較高比例的分裂指數(shù),容易計數(shù),可對原始損傷作出較準(zhǔn)確的估計。 2早熟染色體凝集技術(shù)可以用于估算低劑量受照者輻射劑量。 3在估算低傳能線密度輻射的吸收劑量時,必須考慮劑量率效應(yīng),應(yīng)根據(jù)輻照情況盡量選擇劑量率相近的劑量-效應(yīng)關(guān)系曲線進(jìn)行估算,從而使結(jié)果更為準(zhǔn)確。 4采用QPCC的方法可以較準(zhǔn)確地估算活體局部受照劑量與份額,可用于局部大劑量電離損傷程度測定。
[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.

【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R144.1

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