RESUMO
Objective To establish a quantitative polymerase chain reaction(PCR)method for the analysis of human-derived SRY DNA in mouse tissues,and to study the tissue distribution of human umbilical cord mesenchymal stem cells(HUCMSCs)in immunodeficient NOG mice after a single intravenous injection.Methods We established a quantitative PCR method for the analysis of human SRY DNA in mouse tissues,and validated the standard curve,linear range,accuracy,precision,and stability.Thirty-six NOG mice(18 male,18 female)were administered 3.5×107 HUCMSCs/kg by single intravenous injection.Six mice were then anesthetized and dissected after blood collection(EDTA anticoagulation)at 6,12,24,and 72 h,and at 1 and 2 weeks,respectively.DNA was extracted from lung,kidney,heart,liver,brain,spinal cord,stomach,small intestine,fat,skin,spleen,testis,uterus,and ovary tissues,and the distribution of HUCMSCs in each tissue was determined by the validated quantitative PCR method for detecting the human-derived SRY gene in mouse tissues.In addition,18 NOG mice(9 male,9 female)were divided into control(n = 6)and treatment groups(n = 12)injected intravenously with 0.9%sodium chloride and 3.5×107 cells/kg,respectively.Acute toxic reactions were observed during the administration period,and four animals were dissected at 72 h and at 2 and 4 weeks after administration to observe the gross organs.Mitochondrial protein expression was detected in paraffin sections of lung tissues by immunohistochemistry to analyze the colonization of HUCMSCs in lung tissues.Results The established RT-qPCR method for human-derived SRY DNA in mouse tissues met the validation criteria for each index.After a single intravenous injection in NOG mice,HUCMSCs were mainly distributed in the lungs and blood within 1 week after administration,with higher concentrations in lung tissues than in blood.The concentrations of HUCMSCs in lung tissue and blood remained relatively stable within 6～24 h and 6～72 h,respectively,and then decreased over time.The distribution of HUCMSCs in other tissues was not measured at all sampling points.The colonization result showed that HUCMSCs were detected in lungs 72 h after intravenous injection,but not at 2 and 4 weeks.No obvious acute toxicity was observed in NOG mice after single intravenous administration of HUCMSCs.Conclusions The above method for analyzing the distribution of HUCMSCs in mouse tissue is reliable and feasible.HUCMSCs were mainly distributed in lung and blood in NOG mice within 1 week after a single intravenous injection,and mainly colonized lung tissue at 72 h.A single intravenous administration of HUCMSCs has a good safety profile.
RESUMO
Objective To investigate the effects of the fibrin-derived peptide Bβ15-42 (FgBβ 15-42) on renal inflammation in acute kidney injury (AKI) induced by renal ischemia reperfusion (IR).Methods SD rats were randomly divided into sham group (the abdominal cavity were closed after separating the renal artery),IRI group (renal arteries of rats were occluded with microvascular clamps for 60 min),negative treated group (rats were injected with 3.6 mg/kg random peptide by tail vein) and FgBβ15-42 treated group (rats were injected with 3.6 mg/kg FgBβ15-42 by tail vein).Rats were sacrificed at 24 h or 48 h after reperfusion.Blood and kidney samples were collected and histological changes and renal function were examed.The mRNA and protein expressions of intercellular cell adhesion molecule-1 (ICAM-1) and interleukin-1β (IL-1β) were examined by immunohistochemistry,real-time PCR and Western blotting.Results Compared with sham group,Scr and BUN were obviously increased in IRI group (all P ＜ 0.05),pathologic changes of kidney were more serious (P ＜ 0.05).Compared with IRI group,in FgBβ15-42 treated group Scr and BUN were obviously decreased (all P ＜ 0.05),the injury of kidney tubulointerstitial was less serious (P ＜ 0.05).Compared with sham group,there was increased ICAM-1 and IL-1β in IRI group (all P ＜ 0.05),and they all peaked at 24 h.After treated with FgBβ15-42,the expression of ICAM-1,IL-1β were significantly decreased in kidneys compared to IRI group (all P ＜ 0.05).The above indexes had no significant differences between negative treated group and IRI group (all P ＞ 0.05).Conclusions FgBβ15-42 can protect kidneys against ischemia reperfusion injury in rats.The mechanism may be associated with down-regulated expressions of ICAM-1 and IL-1 β in the kidney.