| 1 ObjectiveMesenchymal stem cells (MSCs) are fibroblast-like pluripotent stem cells that can differentiate into multiple lineages. Initially discovered from bone marrow, MSCs were later isolated from various tissues, including umbilical cord, umbilical cord blood, placenta, fat, muscle, skin, periodontal, etc. Nowaday, it is reported that MSCs can be isolated from almost all adult tissues and organs. Studies show that MSCs can adjust immune function by direct contact or secreted cytokines. As an important part of tumor stroma, MSCs can regulate local immunity and influence proliferation, migration and apoptosis of tumor cells. Some researches show that MSCs have the capacity of promoting tumor growth and metastasis. Meanwhile, another researches show that MSCs can suppress tumor growth and metastasis. The biological functions of MSCs and tumor cells can influence each other. It is even report that MSCs may undergo malignant transformation and convert to tumor stem cells. It isn’t known still whether MSCs exist in renal cell carcinoma, whether the biological functions of MSCs is changed, and whether renal cell carcinoma cells are influenced by MSCs. Astragalus has inhibitory effect on renal cell carcinoma, but the mechanism is unclear. MSCs may be the middle link between Astragalus and renal carcinoma. In this study, we isolate MSCs from normal renal tissues and renal cell carcinoma tissues, compare the biological functions of the two kinds of MSCs, observe the abilities of MSCs to influence migration and proliferation of tumor cells, and research the molecular mechanisms. In addition, we study the effect of Astragalus on the biological characteristics of MSCs. The ultimate goal of this study is seeking new way to treat renal cell carcinoma.2 Methods(1) Isolation and identification of MSCs from normal renal tissues and renal cell carcinoma tissuesSeven normal renal tissues and eight renal cell carcinoma tissues were obtained and were enzymatically dissociated in collagenase and pancreatin. The suspension was cultured in alpha minimum essential medium (a-MEM) containing 10% fetal bovine serum (FBS). MSCs were isolated and cultured by adherence screening method. The adherent fibroblast-like cells were further incubated until 80% confluence.Through the following aspects to observe the biological functions of each generation of MSCs, including the morphology in microscopy, the proliferation curve of MSCs by CCK-8 method, the cell cycle of MSCs by PI staining, the cell surface markers (CD 105, CD73, CD90, CD45, CD34, HLADR) by FACS analysis, the osteogenic diff-erentiation capacity and the adipogenic differentiation capacity.(2) Mesenchymal stem cells promote migration and proliferation of renal cell carcinoma cellsThe renal clear cell carcinoma line (786-0) was divided into three groups:control group, kidney normal MSCs (KN-MSCs) group and kidney cancer-associated MSCs (KC-MSCs) group. The migration rate of three groups was measured by wound healing assay. Control group were incubated in the complete medium (90%RPMI1640+10%FBS). KN-MSCs group were incubated in the complete medium containing supernatants obtained from KN-MSCs. KC-MSCs group were incubated in the complete medium containing supernatants obtained from KC-MSCs.The renal clear cell carcinoma line (786-0) was divided into three groups:control group, kidney normal MSCs (KN-MSCs) group and kidney cancer-associated MSCs (KC-MSCs) group. The proliferation curve of three groups was measured by CCK-8 method. Control group were incubated in the complete medium (90%RPMI1640+10%FBS). KN-MSCs group were incubated in the complete medium containing supernatants obtained from KN-MSCs. KC-MSCs group were incubated in the complete medium containing supernatants obtained from KC-MSCs.Nude mice were divided into three groups:control group, kidney normal MSCs (KN-MSCs) group and kidney cancer-associated MSCs (KC-MSCs) group. Volume and weight of the subcutaneously transplanted tumer were measured. Control group were subcutaneously injected with renal clear cell carcinoma line (786-0). KN-MSCs group were subcutaneously injected with renal clear cell carcinoma line (786-O) and KN-MSCs. KC-MSCs group were subcutaneously injected with renal clear cell carcinoma line (786-O) and KC-MSCs.The total RNA was isolated from KN-MSCs and KC-MSCs, then reverse transcribed. Cytokines (IL-8, CCL5, TNF-α, SDF-1, VEGF, PDGF, FGF, EGF, ICAM-1, N-Cadherin) were detected with Realtime-QPCR.The KN-MSCs was divided into two groups:control group and experiment group. The total RNA was isolated from control group and experiment group, then reverse transcribed. Cytokines (IL-8 and PDGF) wre detected with Realtime-QPCR. Control group were incubated in the MSCs complete medium (90%α-MEM+10%FBS). Experiment group were incubated in the MSCs complete medium containing supernatants obtained from renal clear cell carcinoma line (786-O).(3) Astragalus affect the biological characteristics of MSCs from normal renal tissues and renal cell carcinoma tissuesMSCs were cultued in complete medium with different density of Astragalus. the biological characteristics of MSCs were observed, including proliferation curve, the osteogenic differentiation capacity and the adipogenic differentiation capacity.MSCs were cultued in complete medium with different density of Astragalus. The total RNA was isolated from each group. Cytokines (IL-8, CCL5, TNF-α, SDF-1, VEGF, PDGF, FGF, EGF, ICAM-1, N-Cadherin) were detected with Realtime-QPCR3 ResultsWe successfully isolated adherent fibroblast-like cells from five normal renal tissues and six renal cell carcinoma tissues. Both the cells have same appearence that don’t even change after ten passages. Both the cell growth curve showed that cell proliferation came to exponential phase in three days and to stationary phase in eleven days after passage culture. Cell cycle analysis to KN-MSCs indicated that 93.78% of the cells were in G0/G1 phase, and to KN-MSCs indicated that 93.96% of the cells were in Go/Gi phase. The cell surface markers of both the cells by FACS analysis showed that the expression rates of CD105, CD73 and CD90 were higher than 95%, and the expression rates of CD45, CD34 and HLADR were lower than 2%. Both the cells have the osteogenic and adipogenic differentiation capacity. The results confirm that MSCs can be obtained successfully from normal renal tissues and renal cell carcinoma tissues.The migration rate measured by wound healing assay show that KC-MSCs group has higher migration capacity than control group and KN-MSCs group. The proliferation curve measured by CCK-8 method show that KC-MSCs group has higher proliferation capacity than control group and KN-MSCs group. The subcutaneously transplanted turner of KC-MSCs group nude mice are biggest and heaviest in three groups.The expression of Cytokines (IL-8, CCL5, TNF-a, SDF-1, VEGF, PDGF, FGF, EGF, ICAM-1, N-Cadherin) detected with Realtime-QPCR show that KC-MSCs have higher expressing IL-8 and PDGF capacity than KN-MSCs.The KN-MSCs incubated in the MSCs complete medium containing supernatants obtained from renal clear cell carcinoma line (786-O) have higher expressing IL-8 and PDGF capacity than incubated in the MSCs complete medium only.After cultured with Astragalus, MSCs show greater multiplication capacity.After cultured with Astragalus, MSCs can barely transdifferentiate into adipocytes or osteocytes.After cultured with Astragalus, MSCs have lower expressing multiple Cytokines including IL-8 and PDGF.4 ConclusionMSCs exist in normal renal tissues and renal cell carcinoma tissues and can be extracted. Results in vitro and in vivo experiment indicated that kidney cancer-associated MSCs can make renal clear cell carcinoma line (786-0) have higher migration and proliferation capacity, perhaps because kidney cancer-associated MSCs have higher expressing IL-8 and PDGF capacity than KN-MSCs. Meanwhile, renal clear cell carcinoma line (786-0) can make MSCs express more IL-8 and PDGF in vitro. In addition, some cytokines, including IL-8 and PDGF, will be expressed less by MSCs cultured with Astragalus.
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