The Experiment And Clinical Research Of Human Umbilical Cord Mesenchymal Stem Cells Treatment Children Refractory Pulmonary Diseases

PartⅠEstablishment of ALI animal model and cultivation and identification of HUC-MSCsObjective: Establishment of stable ALI animal model is prepare for HUC-MSCs treatment ALI.To establish a stable and reliable cell batches,culture,identification procedures,for the next step of pre-clinical and clinical research laid the foundation.Methods: C57BL/6 mice(SPF)were randomly divided into normal control group(n=24)and ALI group(n=24).The ALI group were given endotracheal intubation after i.p 10% chloral hydrate(3ml/kg)and endotracheal drip into the LPS 30μl(5.0 mg/kg)solution.The control group were endotracheal drip of PBS 30μl.Then observe the changes of mouse weight and survival.On day 1,3,5 mouse were sacrifice and theW/D ratio of lung tissue,lung histopathologic scores were observed.The levels of total protein,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)in bronchoalveolar lavage fluid(BALF)were measured.With adherent culture method to cultivate batches HUC-MSC.According to the ratio of 2:1 subculture,every three days and an half quantity change culture.The surface antigen CD73,CD105 and CD90,CD34,CD45,HLA-DR expression of P4 generation HUC-MSCs cell were detected by flow cytometry instrument.48 hours after P4 generation HUC-MSCs anchorage growth,giving umbilical cord mesenchymal stem cells induced osteoblast culture,into fat cells induced culture,into cartilage cells culture to observe the differentiation ability into fat,into cartilage and into bone.And identification with alizarin red staining,oil red O staining,the new blue staining.Results: The model group mice began to eat marked less on the next day,and the activity,response to external stimuli,and weight reduced,then the mortality and W/D of lung tissue is higher when compared with normal.Lung gross observation of model group was obviously hyperemia,swelling,surface visible bleeder,then consolidation of the lung tissue was changed,visible: pulmonary interstitial broadening,a large number of inflammatory cells infiltration,alveolar cavity with exudate,histopathologic score significantly higher(P < 0.05);In the model group the levels of totalprotein,TNF-α,IL-6 and IL-10 in BALF obviously increased(P < 0.05).Flow cytometry instrument detection P4 generation of HUC-MSCs cell surface antigen CD73,CD105 and CD90,CD34,CD45,HLA-DR expression rate were 99.97%,99.89%,99.81%,0.14%,0.17%,0.08%,and conformed to the characteristics of HUC-MSCs cell surface markers.Osteogenic induction: Cultured 14 days,and then with alizarin red stain,white calcium nodules were rust red coloring,the control group did not see any color.Adipogenic induction: Cultured a week,the visible with lipid droplets in cytoplasm,train 14 consecutive days,oil red O staining,lipid droplets dyed red.Cartilage induction: Cell ensemble to be suspended in a culture to carry on the induction training,continuous culture around,o line new blue staining,cartilage was dyed blue.Conclusion: The ALI model can be established by driping into the LPS endotracheally.Adherent cell culture method is ideal for training,and extending the HUC-MSCs.Part Ⅱ HUC-MSCs treatment acute lung injury modelObjective: To explore the safety and curative effect of HUC-MSCs for ALI model.Method: We divide 96 C57BL/6 into 4 groups(n=24).After anesthetized with 3ml choral hydrate intraperitoneal injection and the endotracheal incubation is performed.Blank control: 30μl PBS was instilled into air way,and 60μl PBS was instilled after 1 hour;Safety group:30μl PBS was instilled into air passage,and 60μl HUC-MSCs(5×105)was instilled after 1 hour;Model group: 30 ul LPS(5mg)was instilled for ALI model,and 60μl PBS was instilled after 1 hour;Treatment group: 30μl LPS(5mg)was instilled for ALI model,and 60μl HUC-MSCs(5×105)was instilled after 1 hour;The weight and survival rate was recorded and the mouse were separately executed in 1、3 and 5days and the samples was collected.We analyze the wet/dry ratio,HE score of lung,and the total protein and TNF-α,IL-6,IL-10 in BALF.Result: Comparation of the TNF-α,IL-6,IL-10 in BALF and the wet/dry(W/D)ratio,HE score of lung in 1,3 and 5 days between safety group and blank group,there is no significant difference.(P >0.05).Compared with model group,the death rate decreased significantly after treatment with HUC-MSCs,the body weight recovered from the 3rd day(P<0.05).Compared with model group,the wet/dry(W/D)ratio is decreased in treatment group at every time point(P<0.05).Compared with model group,the pneumonedema neutrephil infiltration and HE score is decreased at every time point(P<0.05).Compared with model group,the the total protein in BALF of treatment group decreased significantly at every time point(P<0.05).Compared with model group,the pro-inflammatory factors TNF-α and IL-6 in BALF of treatment group decreased(P<0.05)and on the contrary the anti-inflammatory factor IL-10 increased in BALF of treatment group(P<0.05).Conclusion:Early administration of HUC-MSCs can effectively treat ALI mice,and can significantly improve the prognosis of mice.There is no adverse reactions were found after HUC-MSCs allograft mice.Part Ⅲ Phase I clinical study of human umbilical cord mesenchymal stem cells in the treatment of ARDS in childrenObjective: To explore the safety of HUC-MSCs in the treatment of ARDS in children.Method: From July 2015 to June 2017,the eligible PARDS patients who admitted to intensive care unit of Chongqing Children’s Hospital and research cooperation hospitals were included in this study.One case of traumatic PARDS was included in this study.After admission,the children were given special care,intensive care,on the basis of correcting the underlying disease,given adequate oxygen therapy,correcting electrolyte disorders and acid-base imbalance,and given mechanical ventilation support treatment.After transplantation of 1×107 HUC-MSC disposable infusion,the blood routine,urine routine,liver and kidney function,electrolyte biochemical test,immune globulin,cell immunity,blood gas analysis,lung CT,record the time,weaning time,vasopressor use and transfer time of ICU days were collected.Results: This child with HUC-MSCs after transplantation had a transient fever(38.6 C),the other from the symptoms and signs,blood biochemical,blood,immune function detection,were not found HUC-MSCs had a negative impact on the children before and after transplantation,chest imaging examination found no pulmonary atelectasis or exacerbation the situation.After 19 months of continuous observation,graft versus host disease(GVHD)and various tumors were not found,and the growth and development were not affected.Conclusion: The treatment of HUC-MSCs transplantation on the patients with traumatic PARDS is safe.The next step is to expand the number of PARDS cases into the study.Part Ⅵ Clinical study of human umbilical cord mesenchymal stem cells in the treatment of refractory lung disease in childrenObjective: To observe safety and efficacy of HUC-MSCs in the treatment of refractory lung disease in children.Method: We enrolled the children who were hospitalized in Chongqing Children’s Hospital from December 2015 to June 2017 and clear diagnosis for BPD,BO,PF children,after failure of conventional treatment,informed consent by the parents agreed and signed,and in accordance with the inclusion criteria of refractory pulmonary disease into this study.8 children were included in the study,according to the condition given 2-3 times HUC-MSCs transplantation(the dose of 1×107/10kg).The general condition and adverse reaction of the patients were closely observed.Blood routine,urine routine,liver and kidney function and biochemical test,immunoglobulin,immune cells,viral hepatitis,syphilis serum markers and HIV antigen antibody,arterial blood gas analysis,lung function,lung CT of these children were respectively collected before and after transplantation.To evaluate the safety and efficacy of HUC-MSCs in the treatment of refractory pulmonary disease in childrenResult: The 8 childrens’ body temperature,respiration,pulse were in the normal range fluctuations during the observation.There were no skin pruritus,muscle pain,headache,blurred vision,vomiting,abdominal pain and other common adverse symptoms after transplantation,and there were no signs of general adverse reactions such as rash or jaundice.No shock,organ failure or death occurred in the children.The urine routine examination before and after transplantation were normal;syphilis,hepatitis B virus serum markers and HIV antigen antibody before and after transplantation in 3 months to detect contrast,there was no abnormal change.There were no significant changes in routine blood routine,liver and kidney function,myocardial enzymes and lactic acid before and after transplantation(P>0.05).There were no significant changes in humoral and cellular immunity before and after transplantation,and there was no significant difference between them(P>0.05).8 patients received HUC-MSCs after transplantation,8 cases of the clinical symptoms in the24 hours after transplantation,arterial blood gas,and it is found that the PO2 is significantly increased,the difference was statistically significant(P<0.05),but after the transplantation of the January review of arterial blood gas PO2 return to the level before transplantation,there was no statistically significant difference(P>0.05);The results of chest CT before and after transplantation showed no significant change except for 1children with PF.The lesions of other BPD and BO children were significantly improved,and the difference of CT score was statistically significant(P<0.05).Conclusion: HUC-MSCs is safe for children with refractory lung disease,and long-term safety needs further observation.At present,our small sample results suggest that HUC-MSCs is effective in some children with refractory lung disease,and further expanded sample and multicenter clinical studies are needed to confirm it.