Study Of Clinical Grade Umbilical Cord Mesenchymal Stem Cells In The Treatment Of Radiation-induced Pulmonary Fibrosis

BackgroundRadiation-induced lung injury is a normal lung injury caused by radiation therapy in thoracic malignancies(lung cancer,esophageal cancer,breast cancer,thymoma,etc.).The American Radiation Oncology Collaborative Group according the different times after radiotherapy divided the radiation lung injury into different classes: acute radiation-induced pneumonitis(ARP)within three month after radiation therapy and radiation-induced pulmonary fibrosis(RPF)appears three month after radiation therapy.At present,using the glucocorticoids,immunosuppressive agents and Chinese medicine in the treatment of radiation-induced lung injury could alleviate the early symptoms,but couldn't repair th e pulmonary fibrosis.So it's really necessary to find an effective treatment for radiationinduced lung injury.Mesenchymal stem cells(MSCs)are adult stem cells with self-renewal and multi-directional differentiation potential,which are widely used in regenerative medicine because they can automatically homing to damaged sites,differentiate into tissue cells,paracrine and so on.In recent years the most widely used MSCs are mainly derived from fat,umbilical cord and bone marrow.Experimental research shows that umbilical cord mesenchymal stem cells(UC-MSCs)have several advantages comparing to others: more convenient in taking out the cells,more viability,less immunogenicity,secretion more cytokines,etc.In recent years,there are many preliminary clinical studies about using the MSCs in the treatment of lung disease(acute respiratory distress syndrome,idiopathic pulmonary fibrosis,bronchiolitis,etc.),these studies did not show any serious adverse events.However,because the sample size of these studies were all very small,its validity need to be further confirmed in Phase II clinical studies.The transplantation methods of MSCs in the treatment of lung disease are mainly includes three ways: intraperitoneal,tracheal,and intravenous injection.Some studies have confirmed that the safety and efficacy using the MSCs by tracheal implantation is better than intravenous injection.Many clinical studies have shown that the implant dose of MSCs between 1 × 106 / kg to 1 × 108 / kg is safe and effective.Although the clinical studies of MSCs in the treatment of acute and chronic lung diseases have achieved initial results,there are no reports of MSCs in the treatment of RPF.Some studies shown that fat-derived MSCs can reduce the degree of fibrosis in rat lung tissue by secreting hepatocyte growth factor(HGF)and prostaglandin-2(PGE-2),and the bone marrow-derived MSCs can increase the survival rate of irradiated mice,from the lung tissue pathology found that the deposition of collagen was decreased,the secretion of TNF-? was increased and the secretion of IL-10 was decreased in MSCs treatment group.So the MSCs have the ability to treat the RPF.However,the premise of using the MSCs in the treatment of RPF is that MSCs or MSCs conditioned medium don't have the proliferative effect on the primary chest tumor.There are many contradictions in the impact of MSCs on tumors.The consensus is that MSCs are closely related to tumor cell proliferation or inhibition associated with tumor types and the ti ssue sources of MSCs.It is necessary to determine the effect of the conditioned medium of the selected grade UC-MSCs or MSCs on lung cancer cells before the clinical trials of RPF.Based on the above studies,in order to learn the safety and efficacy of U C-MSCs in the treatment of RPF.Firstly,we observe the proliferative effect on the primary chest tumor in cellular levels.Secondly,we learn the safety and efficacy of UC-MSCs in the treatment of RPF in clinical levels.At last,to provide the theoretical basis for further clinical research.Objective1.To learn the proliferative effect on the chest tumor.2.To learn the safety and efficacy of UC-MSCs in the treatment of RPF.3.To observe the changes of the IL-6,IL-8,IL-10,TGF-?1 and other cytokines in serum and bronchoalveolar lavage fluid(BALF)before and after using the UC-MSCs in the treatment of RPF.Contents1.The proliferative effect on the chest tumor cell of the UC-MSCs.1.1 The proliferative effect on the chest tumor of the UC-MSCs conditioned medium.Using the 12 h,24h,48 h UC-MSCs conditioned medium to co-culture with H446 and H157 cells respectly for 6 days.Using the MTT,CCK and cell count to detect the proliferation of the two tumor cells at the same time every day.1.2 The proliferative effect on the H446 cell of the UC-MSCs.Using the P5-P10 UC-MSCs to co-culture with H446 cells in the proportin of 1:1,2:1,3:1 for 6 days.The proliferation of tumor cells was detected by Incu Cyte software every 1 hour.2.Clinical observation of the treatment of RPF using the UC-MSCs2.1 The safety in the treatment of RPF using the UC-MSCsFor the subject patients,to collect the peripheral blood,to test the complete bloo d count,liver and kidney function in before implantation of the UC-MSCs and 3 days,3 month,6 month after implantation.And observe the skin allergies,cardiovascular and other adverse events before and after implantation of UC-MSCs.2.2 The efficacy in the treatment of RPF using the UC-MSCsTo do the six minute walk distance(6MWD),St George's Respiratory Questionnaire(SGRQ),pulmonary function(FEV1,DLCO,RV),and the chest CT examination and observe the clinical symptoms in before implantation of the UC-MSCs,3 month and 6 month after implantation.2.3 The changes of the cytokines in the treatment of RPF using the UC-MSCsTo collect the peripheral blood before implantation of UC-MSCs and measure the levels of IL-6,IL-8 and IL 10,IL-12,TNF-?,TGF-?1,mysterious motorist program-1(MMP-1),matrix metalloproteinase-7(MMP-7)et al in the serum.Collect the BALF,then inject the UC-MSCs(dose 1 × 106 / kg)into the site of RPF after lavage by the fiberoptic bronchoscopy,and collect the BALF,and test the above cytokines again.To test the above blood indicators in serum in 3 days and 3 month after UC-MSCs implantation.And to Test the above blood indicators in serum and BALF in 3 days and 3 month after UC-MSCs implantation.Results1.The proliferative effect on the chest tumor cell of the UC-MSCs.1.1 The proliferative effect on the chest tumor of the UC-MSCs conditioned medium.The proliferation of H446 cells and H157 cells was not significantly affected b y UC-MSCs conditioned medium in the first 5 days of co-culture.12 h UC-MSCs conditioned medium promoted the proliferation of H446 cells and H157 cells in the 6th day of co-culture.The 48 h UC-MSCs conditioned medium can inhibit the proliferation of cells.The degree of inhibit of 48 h UC-MSCs conditioned medium on H446 cells and H157 cells may be positively correlated with the co-culture time.1.2 The proliferative effect on the H446 cell of the UC-MSCs.UC-MSCs were co-cultured with H446 cells don't promoted the proliferation of H446 cells in.different proportin,and UC-MSCs inhibited the proliferation of H446 cells under the condition of 1:1 cell proportion.2.Clinical observation of the treatment of RPF using the UC-MSCs2.1 The safety in the treatment of RPF using the UC-MSCsThere were no significant different before and after treatment including: the complete blood count(erythrocyte,hemoglobin,white blood cells,platelets),liver function(transaminase),renal function(creatinine,urea nitrogen,urea),(P>0.05).There were no rash and cardiovascular events before and after implantation of UC-MSCs2.2 The efficacy in the treatment of RPF using the UC-MSCs2.2.1 In the 8 patients in the study,the symptoms of spontaneous shortness of breath and cough in 6 patients were gradually improved.2.2.2 CT lung density decreased significantly in 3 days and 3 month after UC-MSCs implantation compared with that before implantation.2.2.3 There were significant differences in SGRQ between before implantation and three month after implantation or six month after implantation(P<0.05),while there were no significant differences in 6MWD(P>0.05).2.2.4 There were no significant difference of the indicators of lung function(FEV1,MVV,DLCO)before and after implantation of UC-MSCs(P> 0.05).2.3The changes of the cytokines in the treatment of RPF using the UC-MSCsTGF-?1 in serum and BALF shown a decreased trend among before implantation and after implantation of the UC-MSCs,but there were no significant difference(P> 0.05).Conclusions:1.Using the 48 h UC-MSCs conditioned medium can inhibit the growth of the H446 and H157 cells.Using the UC-MSCs to co-culture with the H446 cells in 1:1 can inhibit the growth of the H446 cells.2.Using the UC-MSCs in the treatment of RPF,we did not find any side effects to the liver,kidney function,bone marrow hematopoietic function.Using the UC-MSCs in the treatment of RPF could reduce the clinical symptoms,improve the patient's St George's breathing difficulties score and CT lung density.3.Using the UC-MSCs in the treatment of RPF could reduce the degree of fibrosis by reducing the formation of TGF-?1.