荣昌猪BMMNCs诱导分化及其BPI表达与抗病性的研究
摘要
疾病给猪生产带来很大的养殖风险并增加防疫治疗成本,而且预防和治疗的大量用药,导致猪肉食品安全受到潜在威胁。从遗传上降低猪的疾病易感性和提高抗病能力,可以很好解决此问题。BPI基因在其他物种已经研究比较多,有希望成为抗病育种的候选基因。荣昌猪作为地方猪品种,具有抗病抗逆性能好的特点。本研究从体外培养诱导分化荣昌猪骨髓单核细胞(Bone Marrow Mononuclear Cells, BMMNCs)来研究荣昌猪中性粒细胞表达杀菌/通透性增强蛋白基因(Bactericidal/Permeability-Increasing Protein, BPI)的规律,为猪抗病育种提供参考。
1猪骨髓单核细胞培养
优化改进并建立了市售排骨分离猪BMMNCs的新方法,将市售排骨分离获得的猪BMMNCs细胞与无菌手术条件取得的肋骨分离的猪BMMNCs细胞,经过台盼蓝染色测定活细胞率,差异不显著。使用市售排骨进行猪BMMNCs培养,为造血干/祖细胞、间充质干细胞、骨髓多能干细胞移植等研究提供了便利。
研究筛选适宜猪BMMNCs的培养条件为：液体培养,使用RPMI-1640培养液,添加15%FBS,培养环境为5%C02,37℃,饱和湿度。虽然采用离体肋骨分离猪BMMNCs,污染机会并没有显著增加,100U/mL双抗即能达到效果。
2猪骨髓单核细胞增殖
采用细胞计数、MTS/PMS法结合的方法探讨猪骨髓单核细胞的增殖,研究了胰岛素、地塞米松、SCF、G-CSF、GM-CSF等因子对骨髓单核细胞增殖的影响,筛选出促进增殖的最佳浓度SCF为30ng/ml, G-CSF为40ng/ml, GM-CSF为80ng/ml, IL-3为100ng/ml, IL-6为150ng/ml。胰岛素与地塞米松对猪BMMNCs增殖促进作用不显著。
3猪骨髓单核细胞分化
采用瑞氏染色、苏丹黑B染色、墨汁吞噬试验、热盐水溶核试验等方法研究不同培养条件下猪中性粒细胞的分化。瑞氏染色、苏丹黑B染色能较好反映分化状态,而墨汁吞噬试验、热盐水融核试验对荣昌猪中性粒细胞的分化展示作用不好结果表明LPS、地塞米松具有显著促进中性粒细胞分化作用,胰岛素、IL-3、IL-4促进中性粒细胞分化作用不显著。体外培养的荣昌猪BMMNCs可以进行中性粒细胞分化,但分化到杆状核和分叶核中性粒细胞阶段的很少见。
改进染色与杂交用载玻片。常规细胞涂片或细胞爬片,不能在同一张载玻片上同时对多个组进行染色或杂交操作,不同载玻片进行操作必然加大操作误差。本研究将载玻片用疏水的蜡划分成12格,在不同格里涂不同组细胞悬液,可以同时对12组细胞染色或杂交操作,保证条件完全一致,效果优于常规方法。该载玻片专利申请号201120301873.8。
4荣昌猪骨髓单核细胞中BPI表达
设计2个mRNA原位杂交探针,5'-CGTGGACACCTTGGGTATGA G-3和5'-TGCTGCTGTTCATCTCAATC-3',在猪早幼中性粒细胞可以检测到杂交信号,说明在早幼中性粒细胞阶段,BPI就开始表达。经过LPS不同时间刺激后,培养BMMNCs中不同阶段的中性粒细胞BPI mRNA原位杂交阳性率上升,说明LPS可以促进BMMNCs中BPI mRNA合成。
5不同SNP基因型的荣昌猪骨髓单核细胞中BPI表达与攻毒试验
利用PCR-SSCP技术对荣昌猪外显子3进行基因型判定,选择不同外显子猪取BMMNCs进行培养并进行BPI mRNA原位杂交,结果表明不同外显子对骨髓单核细胞中BPI表达影响不显著,但在LPS刺激下,GG基因型的BMMNCs细胞中不同分化阶段的粒细胞中阳性率上升程度高于AA基因型,并且在攻毒试验中,GG和AA基因型均未表现出对致病性大肠杆菌的具有抵抗作用,但GG基因型个体症状较轻,恢复时间短于AA基因型个体。
Diseases lead to great economic risk and increase the cost of treatment and epidemic prevention in pig production, and the medicine widely used for epidemic prevention and treatment of disease may cause potential menace to pork food safety. Reducing disease susceptibility and improving the disease resistance of pig in genetic background would be a suitable solution. BPI gene in many species has been researched intensively for it can serve as candidate gene for disease resistance breeding. As a local pig variety, Rongchang pig has been characterized with high disease resistance. This study dwell on BPI expression in Rongchang pig bone marrow mononuclear cells (BMMNCs)in vitro culture to discuss the possibility of application in pig disease resistance breeding procedure.
1 Culture of Pig Bone Marrow Mononuclear Cells
This research established a new method to separate pig BMMNCs from the resale ribs. The living cell rates of BMMNCs obtained from resale ribs and ribs got from surgical method under sterile condition were compared by means of Trypan blue stain, the result shows that they have no significant difference.The new method brings convenience for the research of the hematopoietic stem/progenitor cells, mesenchymal stem cells and bone marrow stem cells transplantation. The cultivation conditions for pig BMMNCs were optimized:liquid culture with RPMI-1640 medium,15% FBS,37℃and 5% CO2, saturated humidity, 100U/mL penicillin and 100U/mL streptomycin.
2 Proliferation of Pig Bone Marrow Mononuclear Cells
By means of cell counting and MTS/PMS method, the proliferation of pig BMMNCs was discussed and insulin, dexamethasone, SCF, G-CSF, GM-CSF were taken into account to clarify their influence on the proliferation of pig BMMNCs. The optimum concentration combination was determined as SCF 30 ng/ml, G-CSF 40 ng/ml, GM-CSF 80 ng/ml, IL-3100 ng/ml, IL-6150 ng/ml。Insulin and dexamethasone have no promotion effect on pig BMMNCs proliferation.
3 Differentiation of Pig Bone Marrow Mononuclear Cells
Wright's stain, Sudan black B stain, ink particles experiment and hot brine nucleus experiment were employed to research the differentiation of pig BMMNCs under different culture condition. Wright's stain and Sudan black B stain can reflect the differentiation of pig BMMNCs well while the other two methods didn't work well. Further results showed that LPS and dexamethasone promoted the differentiation towards neutrophils significantly while insulin,IL-3, IL-4 did not. The Rongchang pig BMMNCs can differentiate towards neutrophils but seldom reached the stage of rod or segmented neutrophil.
This research designed a new microslide for cell dyeing and hybridizing. In conventional procedure, the cells of each group of a multi-group experiment was smeared or cultured on one microslide and was handled on one's own in the dyeing and hybridizing procedure. Operate respectively could lead to more random errors and conceal the effect of the main factor of the experiment. The new microslide was divided into 12 grids with waxed line. With different group of cells smeared to different grid,12 groups of cells can be handled for dyeing or hybridizing simultaneously. Dyeing or hybridizing with these microslides will result in better consistency than common ones. The patent application number of the microslide is 201120301873.8.
4 BPI Expression in Rongchang Pig BMMNCs
Two in situ hybridization probes were designed to detect BPI mRNA:5'CGTGG ACACCTTGGGTATGAG-3'and 5'-TGCTGCTGTTCATCTCAATC-3'. It was confirmed that BPI mRNA first appeared in promyelocyte neutrophils of the pig BMMNCs. After induction of LPS, the positive rate of in situ hybridization of BPI mRNA in BMMNCs in vitro showed linear increase with the LPS concentration. It means LPS can promote BPI mRNA expression. The positive rate increased in Promyelocyte, Myelocyte and Metamyelocyte, while rod or segmented neutrophils were too rare to reach a statistic result.
5 Impact of Exon 3 Genotypes on BPI expression and Disease Resistance
With PCR-SSCP, we detected the genotype of BPI exon 3 of Rongchang pig and obtained BMMNCs with different genotype to be tested. Result showed that GG type and AA type BMMNCs had no difference in positive rate of BPI mRNA ISH. But after 24 hours induction of 150 ng/ml LPS, GG type BMMNCs had higher ISH positive rate than AA type and control group. In Enteropathogenic E coli challenge test, individuals with GG type and AA type suffered the disease in the same way. But GG type individuals recovered earlier than AA type individuals.
1猪骨髓单核细胞培养
优化改进并建立了市售排骨分离猪BMMNCs的新方法,将市售排骨分离获得的猪BMMNCs细胞与无菌手术条件取得的肋骨分离的猪BMMNCs细胞,经过台盼蓝染色测定活细胞率,差异不显著。使用市售排骨进行猪BMMNCs培养,为造血干/祖细胞、间充质干细胞、骨髓多能干细胞移植等研究提供了便利。
研究筛选适宜猪BMMNCs的培养条件为：液体培养,使用RPMI-1640培养液,添加15%FBS,培养环境为5%C02,37℃,饱和湿度。虽然采用离体肋骨分离猪BMMNCs,污染机会并没有显著增加,100U/mL双抗即能达到效果。
2猪骨髓单核细胞增殖
采用细胞计数、MTS/PMS法结合的方法探讨猪骨髓单核细胞的增殖,研究了胰岛素、地塞米松、SCF、G-CSF、GM-CSF等因子对骨髓单核细胞增殖的影响,筛选出促进增殖的最佳浓度SCF为30ng/ml, G-CSF为40ng/ml, GM-CSF为80ng/ml, IL-3为100ng/ml, IL-6为150ng/ml。胰岛素与地塞米松对猪BMMNCs增殖促进作用不显著。
3猪骨髓单核细胞分化
采用瑞氏染色、苏丹黑B染色、墨汁吞噬试验、热盐水溶核试验等方法研究不同培养条件下猪中性粒细胞的分化。瑞氏染色、苏丹黑B染色能较好反映分化状态,而墨汁吞噬试验、热盐水融核试验对荣昌猪中性粒细胞的分化展示作用不好结果表明LPS、地塞米松具有显著促进中性粒细胞分化作用,胰岛素、IL-3、IL-4促进中性粒细胞分化作用不显著。体外培养的荣昌猪BMMNCs可以进行中性粒细胞分化,但分化到杆状核和分叶核中性粒细胞阶段的很少见。
改进染色与杂交用载玻片。常规细胞涂片或细胞爬片,不能在同一张载玻片上同时对多个组进行染色或杂交操作,不同载玻片进行操作必然加大操作误差。本研究将载玻片用疏水的蜡划分成12格,在不同格里涂不同组细胞悬液,可以同时对12组细胞染色或杂交操作,保证条件完全一致,效果优于常规方法。该载玻片专利申请号201120301873.8。
4荣昌猪骨髓单核细胞中BPI表达
设计2个mRNA原位杂交探针,5'-CGTGGACACCTTGGGTATGA G-3和5'-TGCTGCTGTTCATCTCAATC-3',在猪早幼中性粒细胞可以检测到杂交信号,说明在早幼中性粒细胞阶段,BPI就开始表达。经过LPS不同时间刺激后,培养BMMNCs中不同阶段的中性粒细胞BPI mRNA原位杂交阳性率上升,说明LPS可以促进BMMNCs中BPI mRNA合成。
5不同SNP基因型的荣昌猪骨髓单核细胞中BPI表达与攻毒试验
利用PCR-SSCP技术对荣昌猪外显子3进行基因型判定,选择不同外显子猪取BMMNCs进行培养并进行BPI mRNA原位杂交,结果表明不同外显子对骨髓单核细胞中BPI表达影响不显著,但在LPS刺激下,GG基因型的BMMNCs细胞中不同分化阶段的粒细胞中阳性率上升程度高于AA基因型,并且在攻毒试验中,GG和AA基因型均未表现出对致病性大肠杆菌的具有抵抗作用,但GG基因型个体症状较轻,恢复时间短于AA基因型个体。
Diseases lead to great economic risk and increase the cost of treatment and epidemic prevention in pig production, and the medicine widely used for epidemic prevention and treatment of disease may cause potential menace to pork food safety. Reducing disease susceptibility and improving the disease resistance of pig in genetic background would be a suitable solution. BPI gene in many species has been researched intensively for it can serve as candidate gene for disease resistance breeding. As a local pig variety, Rongchang pig has been characterized with high disease resistance. This study dwell on BPI expression in Rongchang pig bone marrow mononuclear cells (BMMNCs)in vitro culture to discuss the possibility of application in pig disease resistance breeding procedure.
1 Culture of Pig Bone Marrow Mononuclear Cells
This research established a new method to separate pig BMMNCs from the resale ribs. The living cell rates of BMMNCs obtained from resale ribs and ribs got from surgical method under sterile condition were compared by means of Trypan blue stain, the result shows that they have no significant difference.The new method brings convenience for the research of the hematopoietic stem/progenitor cells, mesenchymal stem cells and bone marrow stem cells transplantation. The cultivation conditions for pig BMMNCs were optimized:liquid culture with RPMI-1640 medium,15% FBS,37℃and 5% CO2, saturated humidity, 100U/mL penicillin and 100U/mL streptomycin.
2 Proliferation of Pig Bone Marrow Mononuclear Cells
By means of cell counting and MTS/PMS method, the proliferation of pig BMMNCs was discussed and insulin, dexamethasone, SCF, G-CSF, GM-CSF were taken into account to clarify their influence on the proliferation of pig BMMNCs. The optimum concentration combination was determined as SCF 30 ng/ml, G-CSF 40 ng/ml, GM-CSF 80 ng/ml, IL-3100 ng/ml, IL-6150 ng/ml。Insulin and dexamethasone have no promotion effect on pig BMMNCs proliferation.
3 Differentiation of Pig Bone Marrow Mononuclear Cells
Wright's stain, Sudan black B stain, ink particles experiment and hot brine nucleus experiment were employed to research the differentiation of pig BMMNCs under different culture condition. Wright's stain and Sudan black B stain can reflect the differentiation of pig BMMNCs well while the other two methods didn't work well. Further results showed that LPS and dexamethasone promoted the differentiation towards neutrophils significantly while insulin,IL-3, IL-4 did not. The Rongchang pig BMMNCs can differentiate towards neutrophils but seldom reached the stage of rod or segmented neutrophil.
This research designed a new microslide for cell dyeing and hybridizing. In conventional procedure, the cells of each group of a multi-group experiment was smeared or cultured on one microslide and was handled on one's own in the dyeing and hybridizing procedure. Operate respectively could lead to more random errors and conceal the effect of the main factor of the experiment. The new microslide was divided into 12 grids with waxed line. With different group of cells smeared to different grid,12 groups of cells can be handled for dyeing or hybridizing simultaneously. Dyeing or hybridizing with these microslides will result in better consistency than common ones. The patent application number of the microslide is 201120301873.8.
4 BPI Expression in Rongchang Pig BMMNCs
Two in situ hybridization probes were designed to detect BPI mRNA:5'CGTGG ACACCTTGGGTATGAG-3'and 5'-TGCTGCTGTTCATCTCAATC-3'. It was confirmed that BPI mRNA first appeared in promyelocyte neutrophils of the pig BMMNCs. After induction of LPS, the positive rate of in situ hybridization of BPI mRNA in BMMNCs in vitro showed linear increase with the LPS concentration. It means LPS can promote BPI mRNA expression. The positive rate increased in Promyelocyte, Myelocyte and Metamyelocyte, while rod or segmented neutrophils were too rare to reach a statistic result.
5 Impact of Exon 3 Genotypes on BPI expression and Disease Resistance
With PCR-SSCP, we detected the genotype of BPI exon 3 of Rongchang pig and obtained BMMNCs with different genotype to be tested. Result showed that GG type and AA type BMMNCs had no difference in positive rate of BPI mRNA ISH. But after 24 hours induction of 150 ng/ml LPS, GG type BMMNCs had higher ISH positive rate than AA type and control group. In Enteropathogenic E coli challenge test, individuals with GG type and AA type suffered the disease in the same way. But GG type individuals recovered earlier than AA type individuals.
引文
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