1. 南昌大学2014级研究生答辩报告会
南昌大学第二附属医院
耳鼻咽喉头颈外科
欢迎老师同学莅临指导

2. The effect on proliferation，maturity and apoptosis of mouse eosinophils by knocking out CCR3 gene
Postgraduate： Jialin Wang
Tutor： Professor Yuehui Liu

3. Contents Introduction 2. Materials and Methods 3. Results
4. Discussion
5. Conclusion

4. Introduction
AR is the most common allergic disease that effects upper respiratory , and there are at least half a billion allergic rhinitis patients in the worldwide .
We still haven't found a cure for this disease, and more effective treatments have yet to be developed.

5. Introduction

6. Introduction

7. Introduction

8. Introduction
The Role of Eosinophils in the Chemokine Network of Allergy

9. Introduction
The CC-chemokine receptor-3(CCR3) is a transmenmbrane protein that constitutes one of the receptors for CC-chemokines.
It is localized to cells of key importance to allergic inflammation including dendritic cells, Th2-lymphocytes, eosinophils, basophils, and mast cells as well as to epithelial, smooth muscle, and neural cells. Chemokines interacting with CCR3 include eotaxin-1,2,and 3,MCP,and RANTES.

10. Materials and Methods

11. Materials and methods Materials Experimental Animal
Normal male BALB/c mice （SPF; 6-8 weeks old）
CCR3 gene Knockout male BALB/c mice （SPF; 6-8 weeks old，constructed by The Jackson Laboratory ）

12. Materials and methods Experimental procedure CCR3-/- BALB/c mice n=6
Nomal BALB/c mice
n=6
CCR3-/- Group
Normal Group
DNA PCR Identification
Isolation and ex vivo culture
of mouse bone marrow-derived
eosinophils
EOS Identification
Q-PCR checking the
expression of
ECP、EPO、MBP mRNA
Observing the
proliferation of
cultured EOS
Checking the
apoptosis of
cultured EOS

13. Materials and methods Bone Marrow Derived Eosinophil Cultures
Collect marrow cells from femur
0-4 days
Culture with
SCF、FLT3-L
5-14 days
Culture with
IL-5
EOS
Identification

14. Statistical analysis
Data sets are analyzed by t-test unless otherwise indicated. (If they are correspond to Normal distribution test and homogeneity of variance analysis.)
Data are shown as Mean ± SEM.
P value < 0.05 is considered statistically significant.

15. Result CCR3-/- mice DNA Identification
DNA PCR Amplification Product Analysis
( Gel Electrophoresis)

16. bmEOS Cultures microscopical observation
Result

17. Result Bone Marrow Derived Eosinophil Proliferation
Days
Mean±SEM（×106）
P Value
Normal
CCR3-/-
2.29±0.07
2.45±0.13 2
2.15±0.07
2.16±0.06 4
2.49±0.05
2.51±0.03 6
2.52±0.04
2.54±0.06 8
4.17±0.05
4.19±0.08 10
7.63±0.11
5.97±0.06
**P<0.01 12
14.47±0.25
9.36±0.14 14
25.29±0.44
13.71±0.12
Bone marrow derived Eosinophil was investigated with Cytometry every 2 days. The Proliferation of EOS was slower in the CCR3-/- group than the Normal group between 8-14days (p<0.01 by t-test analysis).

18. Result Bone Marrow Derived Eosinophil Identification CCR3-/- EOS
Normal EOS
Wright’s Stain
(Morphology Identification)

19. Flow Cytometry analysis of Eos
Result
Bone Marrow Derived Eosinophil Identification
CCR3-/- EOS
Normal EOS
Flow Cytometry analysis of Eos
surface proteins
Siglec-F

20. The percentage of Siglec-F
Result
Bone Marrow Derived Eosinophil Identification
Group
Experiment
Sample
Siglec-F+%
Average Siglec-F+%
Mean±SEM%
P Value
CCR3 1 i
91.73%
91.33%
91.31±0.96%
P>0.05 ii
93.38%
iii
88.87% 2
93.03%
92.96%
93.56%
92.29% 3
90.23%
89.63%
85.20%
93.47%
Normal
90.68%
90.04%
90.95±0.47%
91.36%
88.08%
92.64%
91.24%
89.92%
91.15%
95.80%
91.58%
87.26%
91.67%
The percentage of Siglec-F
expression is similar within the CCR3-/- group and the Normal group (p>0.05 by t-test analysis).

21. Result Q-PCR analyze bmEos granule protein mRNA expression
Mean±SEM
P Value
CCR3-/-
Normal
ECP
0.146±0.001
1.000±0.008
***P<0.0001
EPO
0.792±0.038
1.002±0.048
*P<0.05
MBP
0.333±0.016
**P<0.001
The relative mRNA expression analysis calculation using a 2-△△Ct method, The expression of ECP、EPO、MBP mRNA were lower in the CCR3-/- group than the Normal group (p<0.05 by t-test analysis).

22. Materials and methods
Flow Cytometry analysis aopotosis of Eos

23. Materials and methods Flow Cytometry analysis aopotosis of Eos
Group
Mean±SEM
P Value
CCR3-/-
Normal
IL-5（+）
10.81%±0.43%
4.73%±0.36%
P<0.001
IL-5（-）
56.81%±1.05%
30.47%±0.94%
The percentage of apoptosis is higher in the CCR3-/- group than the Normal group (p<0.001 by t-test analysis).

24. Discussion

25. Discussion

26. Discussion EOS inflammation and tissue damage↓
EOS granule protein mRNA expression↓
EOS inflammation and tissue damage↓
EOS maturation↓
AR relieved
CCR3 gene knockout

27. Discussion

28. Discussion EOS inflammation and tissue damage↓
EOS proliferation↓，apoptosis↑
Tissue EOS ↓
AR relieved
CCR3 gene knockout

29. Conclusion
1、Through the culture system in vitro, the highly purified and eumorphism eosinophils were successfully cultured.
2、 Knocking out CCR3 gene does not affect the morphous of bmEOS, but it can reduce the expression of ECP, EPO and MBP mRNA.
3、 Knocking out CCR3 gene inhibits EOS proliferation and promotes EOS apoptosis.
4、Knocking out CCR3 gene is totally blocking the Eotaxin/CCR3 pathway, which inhibits the maturation, proliferation and infiltration process of EOS, could relieves allergic inflammatory.

30. 致谢 三年短暂的硕士研究生学习即将结束，首先非常感谢我的导师刘月辉教授硕士在读期间对我的临床学习、课题研究方面的指导以及生活上的帮助。
衷心感谢朱新华教授在临床学习和课题研究方面给予的指导和帮助！
衷心感谢科室各位老师及同学的帮助和支持！
衷心感谢各位专家能抽出宝贵的时间参加这次答辩会，给我的论文提供了建设性的意见！
衷心感谢父母、朋友对我学习和生活的支持!

31. Thank You !