重组基因工程乳酸菌对肾性高血压犬的降血压作用
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摘要
目的：观察利用改良两肾一夹法制作犬肾性高血压动物模型的效果,研究具有降压肽活性基因工程乳酸杆菌的培养及其生长曲线的观察,检测基因工程乳酸菌对肾性高血压犬的降压效果及其与普通乳酸菌的作用的比较。
方法：选用普通家犬3只,随机分为3组,并分别标记,A、B组行两肾一夹肾性高血压模型手术,C组为假手术组作为阴性对照,并检测各组犬的血压变化。由吉林农业大学馈赠的携带能表达ACE抑制肽外源基因的重组基因工程乳酸菌及带有空载体的乳酸杆菌,利用MRS培养基分别对其进行培养,促进重组基因工程乳酸菌中ACE抑制肽基因的表达,测定其生长曲线、进行菌落计数、根据犬体表面积计算出有效喂服量,并把基因工程菌喂服A、C组犬,空载体菌给B组犬喂服,每日监测各组犬的血压,比较给药前及给药后的血压变化及给药后各组犬的血压变化。
结果：行肾性高血压模型制作手术3周后,A、B两组犬血压开始升高,5周后达到高血压状态,A组与术前相比收缩压升高59mmHg±5mmHg,舒张压升高33mmHg±3mmHg,血压升高达46%；B组与术前相比收缩压升高63mmHg±5mmHg,舒张压升高42mmHg±3 mmHg,血压升高达47%；而作为假手术的C组血压与手术前相比无明显差异。5周末手术组收缩压维持在180-190mmHg,并趋于稳定。
给A、B、C组犬分别喂服工程菌和空载体菌3天后,A、B两组犬血压较给药前均有所下降,但下降幅度较小,收缩压下降1 OmmHg左右,舒张压下降6mmHg左右,无统计学意义(P>0.05),且A、B两组犬血压下降幅度相比较无显著差异(P>0.05),而C组犬血压无变化(P>0.05)。继续按此剂量喂服一周,血压不能继续下降。进一步增加剂量至2倍,继续喂服一周,各组犬血压稳定,无明显变化。
讨论：本研究应用自制的铁丝(直径1.2mm)制作犬肾性高血压模型,术后5周血压升高达46%以上,且血压较稳定,模型制作较成功,说明此改良双肾一夹法对制作犬肾性高血压模型也是可行的,且具有较好的效果。
给肾性高血压犬喂服能表达降压肽的重组基因工程菌,20天后肾性高血压犬血压无明显下降(P>0.05)。分析可能出现此种结果的原因有三点：(1)犬的胃肠解剖生理特性如胃肠液pH值和胃排空时间与人以及鼠均有较大的差别,且其小肠转运时间较短,胃肠道搅拌强度和机械破坏力较大,均不利于乳酸杆菌的粘附及定植。(2)重组基因工程乳酸菌对其所携带的外源基因的表达受很多条件的影响,所以基因工程菌是否能表达ACE抑制肽以及所表达的量还需要进一步对表达的ACE抑制肽进行体外检测。(3)由于每组犬仅有一只,标本例数较少,尽管实验结果为阴性,但不能说明重组基因工程乳酸菌对肾性高血压无降血压效果,所以还需要进一步扩大例数来验证其降血压作用。
结论：通过实验结果可得出如下结论：(1)应用自制的铁丝(直径1.2mm)行改良双肾一夹法制作犬肾性高血压模型是可行的,可获得稳定的肾性高血压模型,具有较好的效果。(2)乳酸杆菌在MRS培养液37℃培养20小时左右,OD值达到1.8时,为发酵最适收获期。(3)由于每组犬的例数较少,未发现重组基因工程乳酸菌对肾性高血压犬的明显降血压作用,降血压效果与普通乳酸杆菌相比较也未发现明显差异,所以还需要进一步扩大例数来验证其降血压作用。
Objective:In order to observe the effect of renal hypertension model in dogs with a modified method produced two kidney one clip。We want to study the culture of the recombinant genetic engineering lactobacillus with antihypertensive activity Peptides observe and its growth curve。In addition, we also should detect the antihypertensive effect of the recombinant genetic engineering lactobacillus in renal hypertensive dogs and compare its effect with the lactic acid bacteria without being recombined.
Method:Three ordinary dogs were choosed, then were randomly divided into 3 groups and marked. We gave A and B groups a two kidneys one clip renal hypertensive model operation, while C group, as a negative control, was not gaven an operation. Then we measured their blood pressure in order to detect the change. The recombinant genetic engineering lactobacillus which can express the foreign gene of ACE inhibitory peptides and lactic acid bacteria with the empty vector, were all from Jilin Agricultural University as a gift. We used MRS medium to train them and promot the recombinant genetic engineering lactobacillus in expression of the ACE inhibitory peptide gene, then we measured the growth curve, carried on the colony counting and counted the effective dosage in dogs according to their body surface area. We fed the dogs of A and C groups with the recombinant genetic engineering lactobacillus, respectively fed the dogs of B group with the empty vector lactobacillus. Wth monitoring of the blood pressure of Dogs daily, we can make a comparison of the change in blood pressure in dogs between before and after the administration of medication and we can also observe the differences of changes in blood pressure of each group by comparison after the administration.
Result:The third week after the renal hypertension model surgery, the blood pressure of A and B groups began to increase. Five weeks later, the blood pressure largely reached the hypertensive state. Compared with the preoperative, A group's systolic blood pressure was 59mmHg±5mmHg higher, and the diastolic blood pressure was 33mmHg±3mmHg. Correspondingly the systolic blood pressure of B group was 63mmHg±5mmHg higher than before, and the diastolic blood pressure 42mmHg±3mmHg higher. The blood pressure of A and B groups relatively elevates the 46% and 47%,while as a sham, the blood pressure of the C group has no significant difference with before. After 5 weeks, the systolic blood pressure of the operation groups remained at 180-190mmHg, and stable.
Feeding the dogs of A, C and B groups respectively with the recombinant genetic engineering lactobacillus and the empty vector lactobacillus for 3 days, the A and B two groups'dogs'blood pressure began to drop, but the decline rate was small.The systolic pressure droped about 10mmHg, and the diastolic pressure droped about 6mmHg. So there has non-statistics significance (P>0.05). Compared with B group,the rate of decline of A group's blood pressure has no remarkable difference (P >0.05), but the dog's blood pressure of C group did not have the change (P>0.05). We continued to feed the dogs clothing one week according to this dosage, but their blood pressure cannot continue to drop. Further we multiplied the dose to 2 times, and continued to feed clothing one week, but each group of dogs'blood pressure was stable, not having obviously changes.
Discussion:In this study, self-made wire (diameter 1.2mm) for canine model of renal hypertension, elevated the blood pressure above 46% after 5 weeks, and the blood pressure was more stable. More successful model shows that this improved method of two kidneys one clip renal canine model of renal hypertension is feasible and has good results.
Feeding renal hypertension dogs with the recombinant genetic engineering lactobacillus which can express the antihypertensive activity Peptides for 20 days, there has no significant blood pressure decrease (P>0.05).We Analysis that such results may be have three reasons:(1) Canine stomach and intestines have anatomical and physiological properties, such as gastrointestinal fluid pH, gastric emptying time, which have large differences with human and mouse. In addition, Canine stomach and intestines have the shorter intestinal transit time and larger gastrointestinal mechanical stirring intensity and destructive power, which are not conducive to the adhesion and colonization of Lactobacillus. (2)The expression of foreign genes carried by recombinant genetic engineering lactobacillus will be affected by many conditions, so whether it can express ACE inhibitory peptides and how much it can express need to take in further detection of ACE inhibitory peptides. (3) Because there is only one dog in each group, which is very few samples, although the results were negative, but it can not explain that the recombinant genetic engineering lactobacillus has not the effect of lowering blood pressure in dogs with renal hypertension. So we need to expand the number of samples to validate it's effect in lowering blood pressure.
Conclusion:According to the results the following conclusions can be drawn:(1) It shows that this improved method of two kidneys one clip canine model of renal hypertension with self-made wire (diameter 1.2mm) is feasible and has good results, with which we can obtain accessible and stable model of renal hypertension. (2) Lactobacillus is cultivated with MRS medium at 37℃for 20 hours, and when OD value reach 1.8, which is the optimum harvest period for the fermentation. (3) Because there is very few samples, we did not find that the recombinant genetic engineering lactobacillus has the effect of lowering blood pressure in dogs with renal hypertension, and we did not find that it's effect in lowering blood pressure has significant differences compared with ordinary lactic acid bacteria. So we need to expand the number of samples to validate it's effect in lowering blood pressure.
方法：选用普通家犬3只,随机分为3组,并分别标记,A、B组行两肾一夹肾性高血压模型手术,C组为假手术组作为阴性对照,并检测各组犬的血压变化。由吉林农业大学馈赠的携带能表达ACE抑制肽外源基因的重组基因工程乳酸菌及带有空载体的乳酸杆菌,利用MRS培养基分别对其进行培养,促进重组基因工程乳酸菌中ACE抑制肽基因的表达,测定其生长曲线、进行菌落计数、根据犬体表面积计算出有效喂服量,并把基因工程菌喂服A、C组犬,空载体菌给B组犬喂服,每日监测各组犬的血压,比较给药前及给药后的血压变化及给药后各组犬的血压变化。
结果：行肾性高血压模型制作手术3周后,A、B两组犬血压开始升高,5周后达到高血压状态,A组与术前相比收缩压升高59mmHg±5mmHg,舒张压升高33mmHg±3mmHg,血压升高达46%；B组与术前相比收缩压升高63mmHg±5mmHg,舒张压升高42mmHg±3 mmHg,血压升高达47%；而作为假手术的C组血压与手术前相比无明显差异。5周末手术组收缩压维持在180-190mmHg,并趋于稳定。
给A、B、C组犬分别喂服工程菌和空载体菌3天后,A、B两组犬血压较给药前均有所下降,但下降幅度较小,收缩压下降1 OmmHg左右,舒张压下降6mmHg左右,无统计学意义(P>0.05),且A、B两组犬血压下降幅度相比较无显著差异(P>0.05),而C组犬血压无变化(P>0.05)。继续按此剂量喂服一周,血压不能继续下降。进一步增加剂量至2倍,继续喂服一周,各组犬血压稳定,无明显变化。
讨论：本研究应用自制的铁丝(直径1.2mm)制作犬肾性高血压模型,术后5周血压升高达46%以上,且血压较稳定,模型制作较成功,说明此改良双肾一夹法对制作犬肾性高血压模型也是可行的,且具有较好的效果。
给肾性高血压犬喂服能表达降压肽的重组基因工程菌,20天后肾性高血压犬血压无明显下降(P>0.05)。分析可能出现此种结果的原因有三点：(1)犬的胃肠解剖生理特性如胃肠液pH值和胃排空时间与人以及鼠均有较大的差别,且其小肠转运时间较短,胃肠道搅拌强度和机械破坏力较大,均不利于乳酸杆菌的粘附及定植。(2)重组基因工程乳酸菌对其所携带的外源基因的表达受很多条件的影响,所以基因工程菌是否能表达ACE抑制肽以及所表达的量还需要进一步对表达的ACE抑制肽进行体外检测。(3)由于每组犬仅有一只,标本例数较少,尽管实验结果为阴性,但不能说明重组基因工程乳酸菌对肾性高血压无降血压效果,所以还需要进一步扩大例数来验证其降血压作用。
结论：通过实验结果可得出如下结论：(1)应用自制的铁丝(直径1.2mm)行改良双肾一夹法制作犬肾性高血压模型是可行的,可获得稳定的肾性高血压模型,具有较好的效果。(2)乳酸杆菌在MRS培养液37℃培养20小时左右,OD值达到1.8时,为发酵最适收获期。(3)由于每组犬的例数较少,未发现重组基因工程乳酸菌对肾性高血压犬的明显降血压作用,降血压效果与普通乳酸杆菌相比较也未发现明显差异,所以还需要进一步扩大例数来验证其降血压作用。
Objective:In order to observe the effect of renal hypertension model in dogs with a modified method produced two kidney one clip。We want to study the culture of the recombinant genetic engineering lactobacillus with antihypertensive activity Peptides observe and its growth curve。In addition, we also should detect the antihypertensive effect of the recombinant genetic engineering lactobacillus in renal hypertensive dogs and compare its effect with the lactic acid bacteria without being recombined.
Method:Three ordinary dogs were choosed, then were randomly divided into 3 groups and marked. We gave A and B groups a two kidneys one clip renal hypertensive model operation, while C group, as a negative control, was not gaven an operation. Then we measured their blood pressure in order to detect the change. The recombinant genetic engineering lactobacillus which can express the foreign gene of ACE inhibitory peptides and lactic acid bacteria with the empty vector, were all from Jilin Agricultural University as a gift. We used MRS medium to train them and promot the recombinant genetic engineering lactobacillus in expression of the ACE inhibitory peptide gene, then we measured the growth curve, carried on the colony counting and counted the effective dosage in dogs according to their body surface area. We fed the dogs of A and C groups with the recombinant genetic engineering lactobacillus, respectively fed the dogs of B group with the empty vector lactobacillus. Wth monitoring of the blood pressure of Dogs daily, we can make a comparison of the change in blood pressure in dogs between before and after the administration of medication and we can also observe the differences of changes in blood pressure of each group by comparison after the administration.
Result:The third week after the renal hypertension model surgery, the blood pressure of A and B groups began to increase. Five weeks later, the blood pressure largely reached the hypertensive state. Compared with the preoperative, A group's systolic blood pressure was 59mmHg±5mmHg higher, and the diastolic blood pressure was 33mmHg±3mmHg. Correspondingly the systolic blood pressure of B group was 63mmHg±5mmHg higher than before, and the diastolic blood pressure 42mmHg±3mmHg higher. The blood pressure of A and B groups relatively elevates the 46% and 47%,while as a sham, the blood pressure of the C group has no significant difference with before. After 5 weeks, the systolic blood pressure of the operation groups remained at 180-190mmHg, and stable.
Feeding the dogs of A, C and B groups respectively with the recombinant genetic engineering lactobacillus and the empty vector lactobacillus for 3 days, the A and B two groups'dogs'blood pressure began to drop, but the decline rate was small.The systolic pressure droped about 10mmHg, and the diastolic pressure droped about 6mmHg. So there has non-statistics significance (P>0.05). Compared with B group,the rate of decline of A group's blood pressure has no remarkable difference (P >0.05), but the dog's blood pressure of C group did not have the change (P>0.05). We continued to feed the dogs clothing one week according to this dosage, but their blood pressure cannot continue to drop. Further we multiplied the dose to 2 times, and continued to feed clothing one week, but each group of dogs'blood pressure was stable, not having obviously changes.
Discussion:In this study, self-made wire (diameter 1.2mm) for canine model of renal hypertension, elevated the blood pressure above 46% after 5 weeks, and the blood pressure was more stable. More successful model shows that this improved method of two kidneys one clip renal canine model of renal hypertension is feasible and has good results.
Feeding renal hypertension dogs with the recombinant genetic engineering lactobacillus which can express the antihypertensive activity Peptides for 20 days, there has no significant blood pressure decrease (P>0.05).We Analysis that such results may be have three reasons:(1) Canine stomach and intestines have anatomical and physiological properties, such as gastrointestinal fluid pH, gastric emptying time, which have large differences with human and mouse. In addition, Canine stomach and intestines have the shorter intestinal transit time and larger gastrointestinal mechanical stirring intensity and destructive power, which are not conducive to the adhesion and colonization of Lactobacillus. (2)The expression of foreign genes carried by recombinant genetic engineering lactobacillus will be affected by many conditions, so whether it can express ACE inhibitory peptides and how much it can express need to take in further detection of ACE inhibitory peptides. (3) Because there is only one dog in each group, which is very few samples, although the results were negative, but it can not explain that the recombinant genetic engineering lactobacillus has not the effect of lowering blood pressure in dogs with renal hypertension. So we need to expand the number of samples to validate it's effect in lowering blood pressure.
Conclusion:According to the results the following conclusions can be drawn:(1) It shows that this improved method of two kidneys one clip canine model of renal hypertension with self-made wire (diameter 1.2mm) is feasible and has good results, with which we can obtain accessible and stable model of renal hypertension. (2) Lactobacillus is cultivated with MRS medium at 37℃for 20 hours, and when OD value reach 1.8, which is the optimum harvest period for the fermentation. (3) Because there is very few samples, we did not find that the recombinant genetic engineering lactobacillus has the effect of lowering blood pressure in dogs with renal hypertension, and we did not find that it's effect in lowering blood pressure has significant differences compared with ordinary lactic acid bacteria. So we need to expand the number of samples to validate it's effect in lowering blood pressure.
引文
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