教案2015-细遗实验chapter4the preparation of healthy adult peripheral bloods common karyotype

1、Cellular and Medical Genetics ExperimentsThe Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Southeast University. Zehui Hong, Ph.D.Association professorChapter 4 The preparation of healthy adult peripheral bloods common karyotypeExperiment Aim Master the skills for c

2、ultivating whole blood and preparing for healthy adult peripheral bloods common karotype.Experiment materilas1. Healthy adult peripheral blood2. Centrifuge tube, centrifuge, glass slide, microscope3. PHA, KCl, Giemsa solution, colchicine liquor, methanol, glacial acetic acid细胞周期Cell cycle间期 Interpha

3、se分裂期 Mitotic phase前期（prophase）中期（metaphase）后期（anaphase）末期（telophase）G1期（first gap phase）S 期（synthetic phase）G2期（second gap phase）Experiment PrincipleThe achroacyte in human peripheral blood is mature immunocyte and remain in G0 period without any generation. PHA is the stimulator of human and other

4、 animals immunocytes caryocinesia, which can change the immunocyte remained in G0 into lympholast and force them to start frequent caryocinesia. Add colchicine when immunocytes have the most frequent caryocinesia to prevent the sprindles from coming into being and make the cells remaining in metapha

5、se period. Then collect the cells and add them in hypotonic buffer to enlarge cells and stretch chromosomes. After that you have to fix the chromosomes and get rid of protein, and debris of RBC by using centrifugation technique. PHAColchicine(CLC)Experiment ProtocalRemove the blood into one 10 ml ce

6、ntrifuge tube. After being balanced, the tubes will be centrifuged at 1000 rpm for 8 min.2. Drop the clear supernatant liquid and remain about 0.5 ml liquid; blow the rest 0.5ml liquid by haustorial tube.3. Add 9ml 0.075mol/L KCl liquor that has been heated before; leave them in water at 37 for 30 m

7、in. (This step is the hypotonicity which is very important for the experiment.)4. Add 1 ml fixation liquid (methanol : glacial acetic acid=3:1). Then centrifuge at 1000 rpm for 8 min.(This step is called prefixation.)5. Drop the clear supernatant liquid and remain about 0.5 ml liquid.6. Add 6 ml fix

8、ation liquid and blow the liquor into suspension. Leave the tube at RT (room temperature) for 30 min. 7. Centrifuge the tube at 1000 rpm for 10 min. 8. Drop the clear supernatant liquid and remain about 0.1-0.2 ml liquid. Them blow the liquid into suspenion.9. Take 1-2 drop of the above suspension a

9、nd drop them on one clean cooled glass slide at the height about 45 cm(15cm). (This step is very important which can help the chromatosomes spread out.)10. Make the glass slide dry in the open air.11. Stain the slides in the Giemsa staining solution (Giemsa stock solution : phosphate buffer=1 : 10)

10、for 10 min. 12. Rinse the slides with water and dry them in the open air.13. Observed the slieds with light microscopeCentrifuged, 1000 rpm, 8 min; Drop the supernatant.+9ml 0.075mol/L KCl, 37 for 30 min in water. hypotonicity+1 ml fixation liquid , Centrifuged, 1000 rpm, 8 min. Drop the supernatant

11、.prefixation+6 ml fixation liquid, RT 30min.Centrifuged, 1000 rpm, 10 min; Drop the supernatant.Dropping slide, Dry.Staining, 10min.Rinse the slides with water and dry.Observed.0.5m100 丹佛 (Denver) 体制(1960)每一条染色体可通过相对长度（Relative length）、臂指数（Arm index）和着丝粒指数（Centromere index）等三个参数予以识别；常染色体按长度递减的次序以122

12、号编号，性染色体则称为X和Y。另外人类的46条染色体应根据长度递减顺序和着丝粒位置划分为7个易区分的组，即以字母AG表示7组染色体，并决定将副缢痕和随体作为识别染色体的辅助指标。 核型分析相对长度=该条染色体长度/单倍染色体全长(含X或Y染色体长度）1000臂指数=长臂长度/短臂长度着丝粒指数=断臂长度/染色体全长染色体绝对长度=放大染色体长度(mm)/放大倍数1000（um)Exercise1. Analysis the prepared healthy adult peripheral achroacytes chromatosmoes and judge the sex of sample supplier.2. What should be taken into consideration to complete the sample perfectly? Think about the experience about own failure and success of the experiment, and division of the chromatosomes according to the Denver System. G带（G banding）染色体核型观察10 40 100 染色体分散情况细胞数目，分裂指数染色体数目染色体形状实验评价染色体分散