Study On The Repairation Of Gene Modified MSCs On Thymus With Radiation Injury

Severe acute radiation sickness (ARS) patients often died of hematopoietic failure,radiation-induced multiple organ failure and secondary serious infection which causedby immune function deficiency.As the technology of hematopoietic stem cellstransplantation is widly used, the patients' hematopoietic function could be recovered;But immune function deficiency and multiple organ failure are still endanger thepatients. The thymus is central immune organs and make the lymphatic progenitorcells which generated by bone marrow differentiate and develop to mature T cells in it.But the thymus is highly sensitive to radiation, it will be injured and the body'simmune function will be affected when ARS happened. Therefore, timely repair ofdamaged thymus can effectively improve the immune function of patients withradiation sickness. Bone marrow mesenchymal stem cells (MSCs) may differentiateinto tissues of mesodermal,ectodemal, and endodermal origin,so MSCs can participatein the repairation of damaged tissue; Meanwhile, MSC can maintain themicroenvironment of thymus matrix and promote T cells mature collaboratively withthymic epithelial cells. Whether MSCs can repair the thymus with radiation injury andrecovery immune function is unknow, thus experiment is study on the problem.ObjectiveThe thymus damaged mouse model which was established after irradiation with60Co γ-rays was used to study on the repairation effect and mechanism of EGFPgenetic modified MSCs.To explore a new way to develope the treatment strategy forsevere ARS.Methods1. Separated and purified C57mice bone marrow MSC by whole bone marrowadherent culture in vitro, then identified the cultured cells by microscope,flowcytometry and directional induction.2. Constructing recombinant adenovirus vector pAdxsi-EGFP with the shuttleplasmid pShuttle-CMV-EGFP and backbone vector pAdxsi, then amplified in H293 cells, determinated the purified virus titer by TCID50. Identified MSC were infectedby adenovirus (pAdxsi-EGFP) with different multiplicity of infection (MOI),infection rate was verified by fluorescence microscope and flow cytometry, Cellcounting kit-8(CCK-8) was used for investigate the influence of vectors on theproliferation of murine MSCs.3. Female BALB/C mice were irradiated with different doses of⁽60_Coγ-rays toestablish thymus damaged model. Mice mediastinum were exposed under three doses:6Gy,9Gy and12Gy, the rest of the site were covered with10cm lead board, doserate is154.7cGy/min, the distance from animal to radioactive sources is80cm, timefor exposure is6Gy:257seconds;9Gy:386seconds;12Gy:514seconds. After theirradiation, the weight, consumption of foodsthff and water, the thymus index, thethymus pathology, esophageal, the trachea and lung of pathology, peripheral blood,the thymus and peripheral T subsets of the mice were measured to judge whether themodel was established successfully and which dose was the best,then establishthymus damage model with the best radiation dose.4. The MSC was transplanted into the mice model though the tail intravenous.Theweight,the thymus index, the thymus pathology, peripheral blood, the thymus andperipheral T subsets, the T cell receptor rearrangement excision circles (TREC), thethymus Sry expression and the IL-4,IFN-γ,TGF-β three kind of cytokine weremeasured to judge the repairation effect and mechanism of MSCs to the radiationinjury of thymus.Results1. The form of second generation MSC under the inverted microscope appears aslong spindle bunches arrangement. MSC highly experssed CD105(69.01%),CD44(95.65%)phenotypes and seldom expressed CD45(0.76%) and CD34(5.37%).Osteogenesis induced3weeks later used the Von Kossa staining and adipogenicinduced2weeks used oil-red-O staining,MSC showed Osteogenic and adipogenicdifferentiation.2. PAdxsi-EGFP restructuring viral vector plasmid cuted into7bands(14kb,11.8kb,3.1kb,2.66kb,2.47kb,1.45kb,0.6kb) by an enzyme Xho I; PAdxsi cutedinto6bands(14kb,11.8kb,4.0kb,2.47kb,1.45kb,0.6kb) by the enzyme Xho I.Titer ofpurified virus were2×10¹⁰pfu/ml. Infection rate was verified by flow cytometry was 7.4%,65.7%,92.3%,94.6%respectively respond to0,50,150,300MOI. MOI for150, cells' form unchanged, but for300MOI, cells' form changed apparently,performance for cell deformation, floating. Growth curve demonstrated no significantdifference between MSC infected and no treatment control(p﹥0.05).3. Compared with the control group,the weight of the radiation exposure groupwere lower, but6Gy group only on the14th day have difference (P <0.05).9Gygroup on the14th and21st day,12Gy on the14th,21st,28th day had differencecompared with control group(P <0.05). On the consumption of foodsthff and water,6Gy and9Gy group had no difference (P>0.05) compared with control group,12Gygroup had difference (P <0.05). Three radiation exposure group were lower thanthose of the control group on the thymus index; compared with the control group,6Gyon the14th,21st,28th day had difference (P <0.05);9and12Gy group on the7th,14th,21st,28th day had difference (P <0.05), the difference is more significantespecially on the21st,28th day (P <0.01). The thymus gland pathology of micemoudle displaied: cortex lymphocytes degeneration necrosis and more cell fragmentsappeared; the thymus small body in the medulla area disappeared, especially on the7th day after irradiation was obvious, the damage degree and the radiation dose werepositively correlated; after the14th day, the thymus gland cells and the thymus glandvolume increased gradually, but on the28th day after irradiation was still not recoverdto normal. The esophageal and trachea pathology of12Gy group revealed mucosabasal cells increased, vascular congestion, the cortex section necrotic fell off, andinflammatory cells appeared in the inherent layer and the submucosa; that means theesophageal and trachea had radiation injury in this group. The white and lymphocytecell counts of peripheral blood in radiation exposure group are lower than those of thecontrol group and it was the lowest on the7th day, then gradually increased, but to the28th day after irradiation was still not completely normal, compared with the controlgroup had significant difference (P <0.01);6Gy and9Gy group had no difference,but12Gy had difference compared with former two on the7th and14th day(P <0.05).The thymus CD4+CD8+cells scale was about70%, CD4+CD8-cells ratio was about20%, CD4-CD8+cells ratio was about7%and the CD4-CD8-cells accounted foronly2%of the control group. After irradiation, CD4+CD8+cells significantlydecreased, the minimum could plunge to a tenth part of nomal, the proportion beganto recover after the14th day. CD4-CD8-cells were increased after irradiation, theproportion could come to the highest on the7th day after exposure, and the amplitude attributes nearly60times, then fell gradually, but the proportion significantly higherthan before even on the28th day after exposure. CD4+CD8-and CD4-CD8+cellsdecreased and coming down to the lowest on the14th, then picked up gradually. TheCD3+cells' proportion of peripheral blood was about40%, CD3+CD4+cells ratiowas about30%, and CD3+CD8+cells ratio was about10%, CD4/CD8about3.0incontrol group. The cells' proportion of the exposed group had a short rise on the1stday after irradiation, then fell down.On the14th day, CD3+cells rebounded, followedby a slow down again; CD3+CD4+, CD3+CD8+cells and the proportion of CD4/CD8continued to decline, but the drop significantly decreased from the14th day. Thethymus and peripheral T subsets of the exposure groups had the same changing trendand had positive relation to radiation dose.4. There was no difference in the indicators between the control group andcontrol+MSC group (P>0.05).Every index of the radiation group had the samechanging trend as the radiation+MSC group except Sry results(trends see the resultsof part3).Compared with the radiation group, the radiation+MSC group was moresuperiority and the differences was significantly.At the same time the radiation+MSCgroup also had difference with control group. Fluorescence pathology revealed thethymus appeard green fluorescence on the1st day after MSC infusion, but onlyaround vessels. As time goes by,the fluorescent gradually permeated to the organ andthe expression reached the peak on the7th day, dissapeard on the35th day. Theconcentration of IFN-γ and TGF-β were increased significantly in the radiation+MSCgroup, but there was no difference between the radiation group and theradiation+MSC group on the concentration of IL-4. The Sry of the radiation+MSCgroup was positive on the14th and28th day, and it is negative in the rest groups.Conclusion1. Cultrued cells were MSC identified by forms, cellular phenotype analysis andmultiplex differentiation capacity test.2. The recombinant adenovirus vector pAdxsi-EGFP was successfully constructed.The gene-modified MSC expressed EGFP stably and proliferation capacity wasn'taffected for150MOI.3. ALL three doses(6Gy,9Gy,12Gy) could damage the mice' thymus.But onlyexposed to9Gy the thymus had a typical change and other organs were nomal. The animal model could be established under the9Gy dose.4. After infusion, gene modified MSC could recruite to the injured thymus andparticipated in the repairation of the thymus, the MSC could also restore the immunefunction effectively.The damaged thymus induced by radiation could be cured bygene modified MSC. MSC is a novel way to cure acute radiation sickness.