Effect On Neural Function And Homing In Different Transplantated Pathways Of HUCBMNCs In Rats With Cerebral Hemorrhage

Objective To explore whether neuron-like cells existed in human umbilical cord bloodmononuclear cells (HUCBMNCs) and to investigate the feasibility of CM-DiI combined withDAPI labeling human umbilical cord blood mononuclear cells. The changes in morphologyand activities of the cells cultured in vitro after double labeling. Methods The isolatedmononuclear cells from30human umbilical cord blood samples were separated.Theexpressions of nestin and neuron-specific enolase (NSE) were observed byimmunofluorescent staining method. The freshly isolated HUCBMNCs were double labeledwith CM-DiI and DAPI. The HUCBMNCs both freshly isolated and double labeled werecultured in vitro.The changes of the morphology were observed under inverted phase contrastmicroscope. The activity of the cells at different times was detected though Trypan bluestaining.At the same time, the HUCBMNCs double labeled by CM-DiI and DAPI werecounted under the inverted fluorescence microscope at different times. Results There were(2.67±1.29)nestin positive cells and (7.37±2.46cells/HP) NSE positive cells in each highpower field(HP,200×)of HUCBMNCs by immunofluorescent staining method. There were3~5/HP neuron-like Cells which were large and with a plurality of coarse long protrusionssimilar to Dendrites and axons at cultured10d-12d. The membrane and nucleus of humanumbilical cord blood cells present red and blue fluorescence respectively under differentwavelengths with fluorescence microscope after double labeling by CM-DiI and DAPI for15minutes. The CM-DiI/DAPI double labeled human umbilical cord blood cells were in vitrocultured for1,3,7,14and21days, and there was no significant difference in the number ofpositive cells double labeled by CM-DiI and DAPI at different time points under fluorescencemicroscope. The survival rates were95.6%-98.8%. There was no significant difference in cellmorphology between the in vitro cultured double labeled cells and the cells withoutlabeling.The growth state, adherent ability and cell proliferation ability of the double labeledcells were not changed. Human umbilical cord blood mononuclear cells can be effectively labeled by CM-DiI and DAPI at the same time.Conclusion There were a few neuron-likecells in HUCBMNCs neural cells could be found in umbilical cord blood mononuclear cells.Both of CM-DiI and DAPI have slower fluorescence decay and non-toxic adverse reactionson the living cells and were suitable for labeling the HUCBMNCs cells.Part Ⅱ Effect on neural function and homing on different pathways oftransplantation of human umbilical cord blood mononuclear cells in rats with cerebralhemorrhageObjective To observe the changes in behavior、the Migration and homing of humanumbilical cord blood mononuclear cells combined to trace by CM-DiI and DAPI which weretransplanted in rats with cerebral hemorrhage by different ways in two weeks and to discussthe best pathway of umbilical cord blood mononuclear cells (HUCBMNCs) transplanted.Methods An animal model of intracerebral hemorrhage(ICH) was established byautologous blood injection/withdraws two times. The success of cerebral hemorrhage modelconfirmed by computed tomography(CT) examination. And then the fresh HUCBMNCswhich were isolated from human umbilical cord blood were doubly marked with CM-DiI andDAPI.were transplanted into experimental animals in vivo respectively by Wister rats, caudalvein, left ventricular and the position of cerebral hemorrhage. The control group was fed wellwithout treatment after modeling successfully and observed the process of natural recovery.The behavior of each model was assessed by Longa5grading method on1,3,7and14days.Each model of the brain was removed through the perfusion method in transplantation of14days. Observation of HUCBMNCs in vivo migration and homing in brain by fluorescencemicroscopy after frozen sections.Results Statistical data suggested that it was a significantdifference in the changes of rats, neurological function at different time points which weretransplanted HUCBMC through the tail vein, left ventricular and cerebral hemorrhage localpathways(F=131.87, P＜0.001). The factors which time and group interaction effect showedthat different treatment tend to the same trends of time(F=35.54, P＞0.05). Influence ofdifferent transplantation methods on changes of neural function in rats was different(F=6.434,P=0.001).The rats, neurological function scores(2.35±0.67) which were transplanted HUCBMC by cerebral hemorrhage local pathways were obviously higher than the othergroups on the1st day. And the scores(0.40±0.60,0.25±0.37,0.03±0.22) were lower than theother groups on the3th,7th and14th days. There were significant differences between thegroup transplanted by the local cerebral group and the other groups on the3th,7th and14thdays(F=5.59,22.94and11.07, All of P＜0.01).There were no significant differences betweenthe group transplanted by caudal vein and left ventricular(P=0.85,0.08,0.70,0.68).Humanumbilical cord blood mononuclear cell transplantation could improve the neural function inrats with cerebral hemorrhage through the tail vein, left ventricular and cerebral hemorrhagelocal pathway. The best way of transplantation was local cerebral transplantation;TheUmbilical cord blood mononuclear cells which were doubly labeled by CM-DiI and DAPIcould be found in the injured brain tissues of rats aftet the three pathways oftransplantation.The number of the cells were7.93±4.184、16.87±3.31、34.87±2.66/HP bythe pathways of Wister rats, caudal vein, left ventricular. Each of the groups were comparedwith LSD-t test.The result showed that all the P <0.01.In short,it suggested that the best wayof transplantation be the local cerebral transplantation after Statistical treatments（F=458.762,P<0.05）.Conclusion The umbilical cord blood mononuclear cells couldhome to the injured brain tissues of rats aftet the three pathways of transplantation. Thelocal cerebral transplantation was the best way.