In vitro labeling and tracing of metanephric mesenchymal cells derived from embryonic rats

Yuqing JIAO; Zhuwen YI; Xiaojie HE; Xihong LIU; Qingnan HE; Danlin HUANG; Shuanghong MO; Weian FU

ABSTRACT

BACKGROUND:

Stem cell transplantation provides a new approach to treat chronic renal disease.Specific marking and in vivo tracing of stern cells are the basis of studies in this field.However,the marking methods appropriate for all cells remain uncertain.

OBJECTIVE:

To observe the in vivo location and differentiation of 4',6-diamidino-2-phenylindole (DAPI) and green fluorescence protein (GFP)-Iabeled cells in adriamycin nephrosis rats so as to explore an efficient labeling and tracing method for metanephric mesenchymal cells (MMCs) derived from embryonic rats.DESIGN,TIME AND

SETTING:

Grouping comparative observation was performed at the Second Xiangya Hospital of Central South University from April to December 2007.MATERIALSA total of 60 female SD rats,weighing 180-220 g,of dean grade,were used to establish models of adriamycin nephrosis.

METHODS:

DAPI and MMCs infected with GFP and DAPI were respectively injected into addamycin nephrosis via the tail vein.DAPI and GFP distribution in the frozen sections was detected at 1,3,and 5 weeks,postoperatively.In addition,GFP expression in renal tissues was detected by ABC immunoenzymatic staining method.MAIN OUTCOME

MEASURES:

DAPI and GFP-labeled Cell grafts in adriamycin nephrosis rats were compared.The changes of GFP-transfected MMCs at different time points were observed.

RESULTS:

DAPI positive cells were observed in tubular structures after 1 weeks of injection of DAPI-labeled cells and DAPI alone,and remained existing at 5 weeks,but the florescence was reduced with time.GFP-transfected MMCs were able to survive and integrate into tubular structures after 1 week,and remained existing at 5 weeks.Moreover,the fluorescence was not reduced.ABC immunoanzymatic staining showed that only a few GFP-positive MMCs appeared in glomerular tufts,and mainly distributed in cytoplasm.Semi-quantitative evaluation of GFP show that the positive cell rate in rats with early application was greater than that with advanced application,and the positive rate was increased with time.

CONCLUSION:

Liposome mediated GFP gene transfer was an efficient labeling in vitro and suitable tracing method for cell differentiation experiment in vivo,suitable for short-term tracing and observation of transplanted cells.