乳酸杆菌基因工程菌肠粘膜粘附以及对小分子尿毒素的分解研究

发布时间：2018-04-13 03:24

本文选题：基因工程菌 + 黏附　；参考：《中南大学》2012年硕士论文

【摘要】：目的： 1.观察乳酸杆菌基因工程菌在体外对人结肠癌类上皮细胞HT-29细胞的黏附能力,以及在5/6肾切模型大鼠肠道粘附情况。 2.观察乳酸杆菌基因工程菌在体外对小分子尿毒素分解情况,以及对5/6肾切模型大鼠血清、肠道小分子尿毒素的降解能力。方法： 1.细菌粘附实验①实验组,为基因工程菌；对照组,为野生菌。常规培养结肠癌HT-29细胞,分别向HT-29细胞加入1ml菌液浓度约1.5×108cfu/ml的两组细菌共孵,在不同时间点(0.5h、1h、2h、4h、8h、24h)油镜下观察两组细菌的黏附情况比较两者的黏附指数。②SD大鼠行5/6肾切除术后分为三组：1)实验组(n=13),基因工程菌1.5×108cfu/ml×2ml/d灌胃1次/天。2)对照组(n=12),野生菌1.5×108cfu/ml×2ml/d灌胃1次/天。3)病理组(n=10)生理盐水2ml/天。4周后处死所有大鼠留取三组老鼠胃、空肠、结肠,刮取各段肠道粘膜,充分匀浆后、涂片,在油镜下观察细菌数,比较三组细菌数的差异。 2.细菌分解小分子尿毒素实验①实验组,为基因工程菌；对照组,为野生乳酸杆菌(野生菌)；病理组,为无处理肾衰患者血清。向两组细菌中加入肾衰患者血清1ml,在不同时间点(0.5h、1h、2h、4h、8h、24h)与病理组比较观察两种细菌分解肌酐、尿素氮、尿酸的差异。②SD大鼠行5/6肾切除术,眼眶静脉采血后分为三组：1)实验组(n=13),基因工程菌1.5×1O8cfu/ml×2ml/d灌胃1次/天。2)对照组(n=12),野生菌1.5×108cfu/ml×2ml/d灌胃1次/天。3)病理组(n=10)生理盐水2ml/天。4周后处死大鼠留静脉血测肌酐、尿素氮和尿酸值。留取三组大鼠胃、空肠、结肠内容物稀释、离心后测肌酐、尿素氮、尿酸值。结果： 1.细菌粘附实验 1)体外实验结果显示,在不同时间点与对照组相比,实验组基因工程菌对结肠癌HT-29细胞的粘附指数未见不同,无统计学差异(P0.05)。 2)体内实验结果显示,与病理组相比,实验组大鼠肠道粘附的乳酸杆菌明显增多(P0.05),其中结肠粘附的细菌数最多。与对照组相比,实验组胃肠道粘附的乳酸杆菌数量无统计学意义(P0.05)。 2.细菌分解小分子尿毒素实验 1)体外实验结果显示,两组细菌在0.5h、1h、2h、4h四个时间点对小分子尿毒素的降解能力与病理组相比无差异(P0.05)。在8h时间点,实验组肌酐下降较快,与病理组相比差异有意义(P0.05)。24h时间点两组细菌中的小分子尿毒素有不同程度的降低,实验组肌酐、尿素氮、尿酸值低于病理组(P0.05),其中肌酐和尿酸值低于对照组(P0.05)。 2)体内实验结果显示,灌胃前三组大鼠血清肌酐、尿素氮、尿酸值无差别(P0.05)。灌胃4周后,实验组和对照组大鼠血清小分子尿毒素水平降低,病理组大鼠血清小分子尿毒素明显升高。实验组与病理组相比肌酐和尿酸值下降具有统计学意义(P0.05)。与对照组相比,实验组血清肌酐、尿素氮、尿酸水平下降幅度分别为36%、7%、30%。实验组大鼠消化道各段内容物小分子毒素水平低于病理组,其中以肌酐和尿酸明显(P0.05)。虽然对照组中肠道小分子尿毒素低于病理组,但与实验组相比,肌酐和尿酸水平仍较高(P0.05)。结论： 1.乳酸杆菌基因工程菌接受肌酐水解酶和尿酸氧化酶基因重组后,在体外粘附HT-29细胞与野生乳酸菌相比无差别,粘附功能无改变。 2.乳酸杆菌基因工程菌在体内粘附胃肠道粘膜的能力与野生乳酸菌无差别。 3.乳酸杆菌基因工程菌降解小分子毒素能力强于野生乳酸杆菌。 4.乳酸杆菌基因工程菌通过降解胃肠道中小分子尿毒素,从而降低血清中的肌酐、尿酸水平。
[Abstract]:Objective:
1. to observe the adhesion ability of Lactobacillus genetic engineering bacteria to human colon cancer HT-29 cells in vitro and the intestinal adhesion in 5/6 nephrectomy rats.
2., we observed the decomposition of small uremic toxins in vitro by Lactobacillus genetic engineering bacteria, as well as the degradation ability of serum and intestinal small molecular toxins in 5/6 nephrectomy rats.
1. bacterial adhesion experiments the experimental group, as control group, gene engineering bacteria; wild bacteria. Cultured colon cancer cells HT-29, two groups of bacteria respectively to HT-29 cells with 1ml concentration of about 1.5 * 108cfu/ml co cultured at different time points (0.5h, 1H, 2h, 4h, 8h, 24h) the adhesion index of adhesion was observed in the two groups of bacteria under microscope respectively. The SD rats underwent 5/6 nephrectomy were divided into three groups: 1) the experimental group (n=13), gene engineering bacteria of 1.5 * 108cfu/ml * 2ml/d gavage 1 times / day).2 control group (n=12), wild mushroom 1.5 * 108cfu/ml * 2ml/d gavage 1 times / day).3 pathology group (n=10) normal saline 2ml/ days after.4 weeks all rats were sacrificed for three groups of mice stomach, jejunum, colon, scraping of intestine mucosa, fullyhomogenized, smear, observe bacteria under the microscope, the difference between the three groups of the number of bacteria.
2. bacterial decomposition of micromolecule urotoxin experiment as the experimental group, control group, gene engineering bacteria; Lactobacillus (wild wild mushroom); pathology group, no treatment for patients with renal failure. Serum was added to patients with renal failure serum 1ml two groups of bacteria, at different time points (0.5h, 1H, 2h, 4H, 8h 24h), compared with the pathological observation of two bacterial decomposition of urea nitrogen, creatinine, uric acid. The difference of SD rats by 5/6 nephrectomy, orbital venous blood were divided into three groups: 1) the experimental group (n=13), gene engineering bacteria of 1.5 * 1O8cfu/ml * 2ml/d gavage 1 times / day).2 control group (n=12), wild mushroom 1.5 * 108cfu/ml * 2ml/d gavage 1 times / day).3 pathology group (n=10) normal saline 2ml/ days after.4 weeks the rats were sacrificed with blood creatinine, urea nitrogen and uric acid. Specimens from three groups of rats stomach, jejunum, colon contents were measured after dilution, centrifugation creatinine, urea nitrogen, uric acid.
Result:
1. bacterial adhesion experiment
1) in vitro results showed that in different time points, the adhesion index of genetically engineered bacteria to colon cancer HT-29 cells in the experimental group was not different from that in the control group, and there was no statistical difference (P0.05).
2) in vivo experiments showed that compared with the pathological group, the Lactobacillus adhered to the intestinal tract of the experimental group increased significantly (P0.05), and the number of bacteria adhered to the colon was the largest. Compared with the control group, the number of lactobacilli adhered to the gastrointestinal tract in the experimental group was not statistically significant (P0.05).
2. bacteria decomposition of small molecular urine toxin
1) in vitro experiments showed that two groups of bacteria in 0.5h, 1H, 2h, 4H four time points for micromolecule urotoxin degradation ability compared with pathological group had no difference (P0.05). At the time point of 8h, the experimental group were significant decreased rapidly, compared with the pathologic group (P0.05) of small molecules in urine two groups of bacteria in toxin.24h are reduced in different degree, the experimental group of creatinine, urea nitrogen, uric acid value is lower than the pathological group (P0.05), the serum creatinine and uric acid were lower than that of control group (P0.05).
2) in vivo experiment results show that, before gavage three groups of rats serum creatinine, urea nitrogen, uric acid had no difference (P0.05). After 4 weeks, the experimental group and the control group of rats serum micromolecule urotoxin levels decreased, the pathological group of rats serum micromolecule urotoxin increased significantly in the experimental group and the. The pathological group compared to creatinine and uric acid values were statistically significant decrease (P0.05). Compared with the control group, experimental group, serum creatinine, urea nitrogen, uric acid levels decline was 36%, 7%, 30%. experimental group rat digestive tract contents of small molecule toxin levels below the pathology group, the serum creatinine and uric acid significantly (P0.05). Although the small intestinal molecular control group urotoxin below pathology group, but compared with the experimental group, creatinine and uric acid level is still high (P0.05).
Conclusion:
1. Lactobacillus genetic engineering bacteria had no difference in adhesion to HT-29 cells in vitro when they were reconstituted by recombinant creatinine hydrolase and urate oxidase gene. There was no difference in adhesion function between them.
The ability of 2. lactobacilli gene engineering bacteria to adhere to the intestinal mucosa in the body is not different from that of the wild lactic acid bacteria.
The ability of 3. lactobacilli gene engineering bacteria to degrade small molecular toxins is stronger than that of wild Lactobacillus.
4. Lactobacillus gene engineering bacteria can reduce the serum creatinine and uric acid level in the serum by degrading small molecular urine toxins in the gastrointestinal tract.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R371

【参考文献】