人脐带间充质干细胞对耐亚胺培南铜绿假单胞菌耐药性形成的影响

doi:10.3877/cma.j.issn.1674-1358.2016.02.025

目的

探讨人脐带间充质干细胞（hUCMSCs）体外对耐亚胺培南铜绿假单胞菌（IRPA）耐药性形成及对OprD2基因的影响。

方法

本实验设立3个组，实验组为hUCMSCs组，对照组为细胞对照组（即人肺成纤维细胞组，NHLF组）和空白对照组。次抑菌浓度肉汤诱导PA耐药传导过程中，hUCMSCs组和NHLF组分别加入其与PA共培育所得的上清液，空白对照组加入细胞培养液，观察3组诱导耐药所需代数以及抑菌圈的大小。诱导耐药前后分别采用K-B法及real-time PCR法测定PA对常见抗菌药物的敏感性及OprD2基因的表达量。

结果

经次抑菌浓度的亚胺培南诱导后，hUCMSCs组PA耐药性的出现较NHLF组及空白对照组延迟。NHLF组和空白对照组PA于诱导的第17代出现亚胺培南耐药，而hUCMSCs组PA于第19代出现耐药性。real-time PCR结果显示，诱导耐药后PA中OprD2表达量较诱导前出现减少或消失。其中hUCMSCs组PA OprD2的表达量减少至诱导耐药前的10.96%，而NHLF及空白对照组OprD2无表达，即诱导后出现OprD2基因缺失。

结论

人脐带间充质干细胞具有延迟PA耐药性形成的作用，其机制可能是通过分泌抗菌肽LL-37和人β防御素-2从而抑制OprD2表达的减少，而外膜蛋白OprD2表达量减少或缺失是引起PA对亚胺培南耐药的原因。

关键词: 铜绿假单胞菌, 耐药性, 人脐带间充质干细胞, 抗菌肽

Objective

To investigate the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro on the development of the drug resistance of imipenem-resistant Pseudomonas aeruginosa and on OprD2 gene.

Methods

This experiment included three groups: experiment group (hUCMSCs group), control group (cells control group, namely normal human lung fibroblast, NHLF group) and blank control group. During the conductive process of Pseudomonas aeruginosa (PA) drug resistance induced by sub-inhibitory concentrations of broth, the hUCMSCs group and the NHLF group were added with the supernatants obtained from their joint cultivation with PA, respectively; while the blank control group was administrated with cell culture medium. The numbers of generations required by induced drug resistance in the three groups and the sizes of inhibition zones were observed. The sensitivity of PA to common antibacterial agents and the expression quantity of OprD2 gene were determined by K-B method and real-time PCR method.

Results

Through the induction of sub-inhibitory concentration of imipenem, the occurrence of PA drug resistance in the hUCMSCs group was relatively later than that of the blank control group. Imipenem drug resistance appeared in passage 17 of PA induced in the NHLF group and the blank control group, while it occurred in passage 19 in the hUCMSCs group. The results of real-time PCR showed that after the induction of drug resistance, the expression quantity of OprD2 in PA decreased or disappeared compared with that before the induction. The expression quantity of OprD2 in PA in the hUCMSCs group was reduced to 10.96% of the induction, while there were no OprD2 expression in the NHLF and the blank control group, OprD2 gene deletion occurred after the induction.

Conclusions

hUCMSCs could delay the formation of PA’s drug resistance. This mechanism is probably through reducing the inhibition of the expression of OprD2 by secreting antimicrobial peptide LL-37 and human β-defensin 2 (HBD-2), and the decrease or deletion of the expression of outer membrane protein OprD2 is the cause for PA’s drug resistance to imipenem.

Key words: Pseudomonas aeruginosa, Drug resistance, Human umbilical cord mesenchymal stem cells (hUCMSCs), Antimicrobial peptides

表1 内参基因与目的基因引物序列

基因	序列	产物大小（bp）
16S RNA	F：TAGGTGGTTCAGCAAGTTGGATGTGAAATC	174
	R：TGTCAGTATCAGTCCAGGTGGTCGC
OprD2	F：ACCTAGCCTCCTATGGCGTTCCC	150
	R：CGAGGTTGGTTTCGTGGTGCTT

表1 内参基因与目的基因引物序列

基因	序列	产物大小（bp）
16S RNA	F：TAGGTGGTTCAGCAAGTTGGATGTGAAATC	174
	R：TGTCAGTATCAGTCCAGGTGGTCGC
OprD2	F：ACCTAGCCTCCTATGGCGTTCCC	150
	R：CGAGGTTGGTTTCGTGGTGCTT

表2 三组共培育后上清液中抗菌肽的水平（±s，ng/ml）

组别	株数	Cathelicidin/LL-37	HBD-2
hUCMSCs实验组	5	5.90 ± 0.51	1.77 ± 0.25
NHLF对照组	5	3.98 ± 0.34^a	0.72 ± 0.25^a
空白对照组	5	3.09 ± 0.20^b	0.63 ± 0.20^b

表2 三组共培育后上清液中抗菌肽的水平（±s，ng/ml）

组别	株数	Cathelicidin/LL-37	HBD-2
hUCMSCs实验组	5	5.90 ± 0.51	1.77 ± 0.25
NHLF对照组	5	3.98 ± 0.34^a	0.72 ± 0.25^a
空白对照组	5	3.09 ± 0.20^b	0.63 ± 0.20^b

表3 诱导耐药前后PA株对常见抗菌药物的抑菌圈大小（mm）

组别	IPM	AK	CAZ	TZP	MEM	ATM
诱导前原始菌株	25	26	25	30	26	27
诱导后hUCMSCs组	13^*	27	26	29	18	25
诱导后NHLF对照组	12^*	26	27	29	13^*	26
诱导后空白对照组	12^*	26	25	28	14^*	25

表3 诱导耐药前后PA株对常见抗菌药物的抑菌圈大小（mm）

组别	IPM	AK	CAZ	TZP	MEM	ATM
诱导前原始菌株	25	26	25	30	26	27
诱导后hUCMSCs组	13^*	27	26	29	18	25
诱导后NHLF对照组	12^*	26	27	29	13^*	26
诱导后空白对照组	12^*	26	25	28	14^*	25

图2 PA总RNA电泳图

图3 内参基因扩增曲线与熔解曲线图

图4 OprD2基因的扩增与熔解曲线图

图5 PA菌株诱导耐药前后OprD2基因表达水平

图1 三组PA诱导传代至P17时对亚胺培南的敏感性比较

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Effect of human umbilical cord mesenchymal stem cells on the development of the drug resistance of imipenem-resistant Pseudomonas aeruginosa

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Effect of human umbilical cord mesenchymal stem cells on the development of the drug resistance of imipenem-resistant Pseudomonas aeruginosa