The Study In Vivo Of Dual-targeted Therapy Using Mesenchymal Stem Cell^TRAIL For Pancreatic Carcinoma

Objective: To establish model of insitu xenograft in nude mice with human pancreatic cancerfluorescence cells ASPC-1-dsRed and construct mesenchymal stem cell (MSC)-TRAIL transgeniccomplex, then noninvasively monitor and analyse the homing ability of MSC-TRAIL as well as itsfunction of tumor inhibition to pancreatic cancer in vivo. This study will provide a betterexperimental basis and platform for fundamental research of biological characteristics of pancreaticcancer in vivo and the birth of new therapeutic strategies.Methods:(1) Human pancreatic cancer cell line ASPC-1was transfected with red fluorescentprotein (RFP)-dsRed gene which will express RFP stably and highly.(2) Subcutaneous pancreatictumor model、matrigel block orthotopic transplantation model and tumor mass orthotopictransplantation model were established. All the three groups for positive rate of tumorinoculation、growth rate of tumor size and metastasis rate in organs were noninvasively observedby fluorescence imaging system in vivo as well as the situation of tumor growth. Then the bestmodel was accepted.(3) MSC derived from human marrow bone of healthy donors was isolatedand purified. Then enhanced green fluorescent protein (eGFP)-TRAIL plasmid was transfectedinto MSC to structure MSC-TRAIL transgenic complex and the efficiency of transfection waschecked.(4) MSC-eGFP transgenic complex was injected into tumor-bearing mouse viaintraperitoneal and intravenous route. The distribution and tropism to pancreatic cancer of MSC invivo was monitored by fluorescence imaging system.(5) MSC-TRAIL transgenic complex wasgiven into tumor-bearing mouse via suitable route. Then we observed its inhibition ability toorthotopic and metastatic tumor and analysed the correlation between the quantity and frequencyof given complex and tumor size comprehensively.Results:(1) Human pancreatic cancer cell ASPC-1-dsRed labeled by RFP displayed uniformred fluorescence under fluorescent microscopy and showed sustainedly stable expression in vitroand in vivo.(2) We established three groups of pancreatic cancer xenografts in nude mousesuccessfully. The positive rate of construction of the three models is as follows:55%(11/20) forsubcutaneous model,100%(20/20) for matrigel block orthotopic transplantation model,100%(20/20) for tumor mass orthotopic transplantation model. And the metastasis of surrounding organsoccurred in the later two models30days later. The rate of tumor formation in the three groups had significant differences (P <0.01), which is slowest in subcutaneous model and fastest in tumormass orthotopic transplantation model.(3) MSC derived from human marrow bone of healthydonors were isolated and purified successfully as well as be transfected by TRAIL-eGFP plasmid.Green fluorescence was observed under fluorescent microscopy and the product expressed wasverified by Western Blot.(4) It is earlier to observe Homing phenomenon of MSC throughintravenous route than intraperitoneal route in15days by fluorescence imaging system, afterinjecting MSC-eGFP transgenic complex into tumor-bearing mouse via intraperitoneal andintravenous route. The tumor volume of intravenous group after36d is510.88±55.96mm3, andThe tumor volume of intraperitoneal group after36d is425.96±46.55mm3, but there is nostatistical differences between the two groups (P>0.05).(5). Average volume of tumors in eachgroup24d after giving drugs: Control:419.19±21.96mm3; MSC:550.22±59.48mm3; LL-MSC-TRAIL:267.65±9.90mm3; LH-MSC-TRAIL:169.96±10.05mm3; HL-MSC-TRAIL:202.99±14.17mm3; HH-MSC-TRAIL:58.21±9.28mm3; There are significant statistical difference among eachgroup (P <0.01).Conclusion: Orthotopic transplantation model of nude mouse established by RFP-labeledhuman pancreatic cancer cell ASPC-1-dsRed can simulate the biological characteristic ofpancreatic cancer in vivo well; MSC has the ability of tropism to pancreatic tissues and canpromote the growth of pancreatic cancer in vivo; MSC-TRAIL transgenic complex can inhibit thegrowth of pancreatic cancer and metastasis effectively in vivo, and there is a positive correlationbetween the quantity and frequency of MSC-TRAIL and the inhibition effect.