本文選題：基因工程菌 + 黏附��； 參考：《中南大學(xué)》2012年碩士論文

【摘要】：目的： 1.觀察乳酸桿菌基因工程菌在體外對(duì)人結(jié)腸癌類上皮細(xì)胞HT-29細(xì)胞的黏附能力,以及在5/6腎切模型大鼠腸道粘附情況。 2.觀察乳酸桿菌基因工程菌在體外對(duì)小分子尿毒素分解情況,以及對(duì)5/6腎切模型大鼠血清、腸道小分子尿毒素的降解能力。方法： 1.細(xì)菌粘附實(shí)驗(yàn)①實(shí)驗(yàn)組,為基因工程菌；對(duì)照組,為野生菌。常規(guī)培養(yǎng)結(jié)腸癌HT-29細(xì)胞,分別向HT-29細(xì)胞加入1ml菌液濃度約1.5×108cfu/ml的兩組細(xì)菌共孵,在不同時(shí)間點(diǎn)(0.5h、1h、2h、4h、8h、24h)油鏡下觀察兩組細(xì)菌的黏附情況比較兩者的黏附指數(shù)。②SD大鼠行5/6腎切除術(shù)后分為三組：1)實(shí)驗(yàn)組(n=13),基因工程菌1.5×108cfu/ml×2ml/d灌胃1次/天。2)對(duì)照組(n=12),野生菌1.5×108cfu/ml×2ml/d灌胃1次/天。3)病理組(n=10)生理鹽水2ml/天。4周后處死所有大鼠留取三組老鼠胃、空腸、結(jié)腸,刮取各段腸道粘膜,充分勻漿后、涂片,在油鏡下觀察細(xì)菌數(shù),比較三組細(xì)菌數(shù)的差異。 2.細(xì)菌分解小分子尿毒素實(shí)驗(yàn)①實(shí)驗(yàn)組,為基因工程菌；對(duì)照組,為野生乳酸桿菌(野生菌)；病理組,為無處理腎衰患者血清。向兩組細(xì)菌中加入腎衰患者血清1ml,在不同時(shí)間點(diǎn)(0.5h、1h、2h、4h、8h、24h)與病理組比較觀察兩種細(xì)菌分解肌酐、尿素氮、尿酸的差異。②SD大鼠行5/6腎切除術(shù),眼眶靜脈采血后分為三組：1)實(shí)驗(yàn)組(n=13),基因工程菌1.5×1O8cfu/ml×2ml/d灌胃1次/天。2)對(duì)照組(n=12),野生菌1.5×108cfu/ml×2ml/d灌胃1次/天。3)病理組(n=10)生理鹽水2ml/天。4周后處死大鼠留靜脈血測(cè)肌酐、尿素氮和尿酸值。留取三組大鼠胃、空腸、結(jié)腸內(nèi)容物稀釋、離心后測(cè)肌酐、尿素氮、尿酸值。 結(jié)果： 1.細(xì)菌粘附實(shí)驗(yàn) 1)體外實(shí)驗(yàn)結(jié)果顯示,在不同時(shí)間點(diǎn)與對(duì)照組相比,實(shí)驗(yàn)組基因工程菌對(duì)結(jié)腸癌HT-29細(xì)胞的粘附指數(shù)未見不同,無統(tǒng)計(jì)學(xué)差異(P0.05)。 2)體內(nèi)實(shí)驗(yàn)結(jié)果顯示,與病理組相比,實(shí)驗(yàn)組大鼠腸道粘附的乳酸桿菌明顯增多(P0.05),其中結(jié)腸粘附的細(xì)菌數(shù)最多。與對(duì)照組相比,實(shí)驗(yàn)組胃腸道粘附的乳酸桿菌數(shù)量無統(tǒng)計(jì)學(xué)意義(P0.05)。 2.細(xì)菌分解小分子尿毒素實(shí)驗(yàn) 1)體外實(shí)驗(yàn)結(jié)果顯示,兩組細(xì)菌在0.5h、1h、2h、4h四個(gè)時(shí)間點(diǎn)對(duì)小分子尿毒素的降解能力與病理組相比無差異(P0.05)。在8h時(shí)間點(diǎn),實(shí)驗(yàn)組肌酐下降較快,與病理組相比差異有意義(P0.05)。24h時(shí)間點(diǎn)兩組細(xì)菌中的小分子尿毒素有不同程度的降低,實(shí)驗(yàn)組肌酐、尿素氮、尿酸值低于病理組(P0.05),其中肌酐和尿酸值低于對(duì)照組(P0.05)。 2)體內(nèi)實(shí)驗(yàn)結(jié)果顯示,灌胃前三組大鼠血清肌酐、尿素氮、尿酸值無差別(P0.05)。灌胃4周后,實(shí)驗(yàn)組和對(duì)照組大鼠血清小分子尿毒素水平降低,病理組大鼠血清小分子尿毒素明顯升高。實(shí)驗(yàn)組與病理組相比肌酐和尿酸值下降具有統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比,實(shí)驗(yàn)組血清肌酐、尿素氮、尿酸水平下降幅度分別為36%、7%、30%。實(shí)驗(yàn)組大鼠消化道各段內(nèi)容物小分子毒素水平低于病理組,其中以肌酐和尿酸明顯(P0.05)。雖然對(duì)照組中腸道小分子尿毒素低于病理組,但與實(shí)驗(yàn)組相比,肌酐和尿酸水平仍較高(P0.05)。 結(jié)論： 1.乳酸桿菌基因工程菌接受肌酐水解酶和尿酸氧化酶基因重組后,在體外粘附HT-29細(xì)胞與野生乳酸菌相比無差別,粘附功能無改變。 2.乳酸桿菌基因工程菌在體內(nèi)粘附胃腸道粘膜的能力與野生乳酸菌無差別。 3.乳酸桿菌基因工程菌降解小分子毒素能力強(qiáng)于野生乳酸桿菌。 4.乳酸桿菌基因工程菌通過降解胃腸道中小分子尿毒素,從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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R371

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