Inkcazo: Iiseli ze-Mesenchymal stem ziyi-subset yeeseli ze-pluripotent ezinokuthi zibekwe zodwa kwi-mesoderm.Ngokuhlaziya kwabo ukuziphindaphinda kunye neempawu zokwahlula-hlula iindlela ezininzi, banamandla aphezulu onyango olwahlukeneyo kwezamayeza.Iiseli ze-Mesenchymal stem zine-phenotype ekhethekileyo ye-immune kunye nekhono lokulawula i-immune.Ke ngoko, iiseli ze-mesenchymal stem sele zisetyenziswa ngokubanzi kwi-stem cell transplantation, ubunjineli bezicubu, kunye nokufakelwa kwamalungu.Kwaye Ngaphaya kwezi zicelo, zisetyenziswa njengesixhobo esifanelekileyo kubunjineli beethishu njengeeseli zembewu kuthotho lovavanyo olusisiseko kunye nolwekliniki.Ukuza kuthi ga ngoku, akukho ndlela yamkelwe ngokubanzi kunye nomgangatho wolawulo lomgangatho weeseli ze-mesenchymal stem.I-Countstar Rigel inokubeka iliso kugxininiso, ukusebenza, kunye neempawu ze-phenotype (kunye neenguqu zabo) ngexesha lokuvelisa kunye nokwahlula kwezi seli ze-stem.I-Countstar Rigel nayo inenzuzo ekufumaneni ulwazi olongezelelweyo lwe-morphological, olunikezelwa yi-brightfield esisigxina kunye nokurekhoda kwemifanekiso esekelwe kwi-fluorescence ngexesha lenkqubo yonke yokuhlola umgangatho weseli.I-Countstar Rigel ibonelela ngendlela ekhawulezayo, ephucukileyo, kunye nethembekileyo yokulawula umgangatho weeseli zesiqu.
Impahla nenkqubo:
Iiseli ze-mesenchymal stem ze-Adipose-derived (AdMSCs) zinikwe nguNjingalwazi uNianmin Qi, isisombululo se-AO / PI (i-Shanghai RuiYu, CF002).I-Antibody: CD29, CD34, CD45, CD56, CD73, CD105, HLADR (BD Inkampani).
Ii-AdMSCs zakhuliswa kwi-37℃, 5% ye-CO2 ye-incubator efumileyo.Gcoba nge-trypsin ngaphambi kokusetyenziswa.
Inkqubo yokungcolisa i-CD marker yalandelwa njengencwadana ye-antibody.
Ubhaqo lwesiphawuli seCD nge-Countstar Rigel:
1. Inkqubo yesicelo sombala wesignali yenziwe ngokuseta itshaneli ye-PE kumfanekiso we-PE fluorescence.
2. Amasimi ama-3 athinjwa kwigumbi ngalinye.
3. Emva kokwenziwa komfanekiso kunye nohlalutyo lokuqala lugqityiwe, i-threshold (log gate) ukusetwa kwe-positive kunye ne-negative transfection yamiselwa yi-FCS software.
Ukulawulwa komgangatho we-stem cell
Lo Mzobo ulandelayo (Umfanekiso 1) ubonisa inkqubo ye unyango lwe-stem cell .
Umzobo 1: Inkqubo yonyango lwe-stem cell
Iziphumo:
Ukumisela ugxininiso, ukusebenza, ukudayamitha, kunye nokudityaniswa kwee-AdMSCs.
Ukusebenza kwee-AdMSCs kwagqitywa yi-AO/PI, Inkqubo yokusetyenziswa kwemibala emibini yenziwa ngokumisela itshaneli eluhlaza kunye nejelo eliBomvu kumfanekiso we-AO kunye ne-PI fluorescence, kunye nentsimi eqaqambileyo.Imizekelo yemifanekiso ibonisiwe kuMfanekiso 2.
Umzobo 2. Imifanekiso yangaphambi kokuthuthwa nasemva kokuthuthwa kwee-AdMSC.A. Ngaphambi kokuthutha;umfanekiso omele ubonisiwe.B. Emva kohambo;umfanekiso omele ubonisiwe.
Ukusebenza kwee-AdMSC kwatshintshwa kakhulu emva kothutho xa kuthelekiswa nangaphambili kwezothutho.Ukuphila phambi kothutho kwakungama-92%, kodwa kwehliswe ukuya kuma-71% emva kothutho.Isiphumo siboniswe kuMfanekiso 3.
Umzobo 3. Iziphumo ezisebenzayo ze-AdMSCs (Ngaphambi kothutho nasemva kothutho)
Idayamitha kunye nokudityaniswa kwagqitywa ngu-Countstar Rigel.Ubungakanani bee-AdMSCs zatshintshwa kakhulu emva kothutho xa kuthelekiswa nangaphambili kwezothutho.Idayamitha phambi kothutho yayiyi-19µm, kodwa inyuke yaya kutsho kwi-21µm emva kothutho.Umdibaniso wangaphambili wezothutho wawuyi-20%, kodwa unyuke waya kuma-25% emva kothutho.Ukususela kwimifanekiso ebanjwe ngu-Countstar Rigel, i-phenotype ye-AdMSCs yatshintshwa ngokukhawuleza emva kokuthutha.Iziphumo ziboniswe kuMfanekiso 4.
Umzobo 4: Ubukhulu kunye neziphumo zokudibanisa.A: Imifanekiso emele ye-AdMSCs, i-phenotype ye-AdMSCs yatshintshwa ngokukhawuleza emva kokuthutha.B: I-aggregation phambi kothutho yayiyi-20%, kodwa inyuke yaya kuma-25% emva kothutho.C: Idayamitha phambi kothutho yayiyi-19µm, kodwa inyuke yaya kutsho kwi-21µm emva kothutho.
Qinisekisa i-immunophenotype ye-AdMSCs ngu-Countstar Rigel
I-immunophenotype ye-AdMSCs igqitywe ngu-Countstar Rigel, i-AdMSCs yafakwa kunye ne-antibody eyahlukileyo ngokulandelanayo (CD29, CD34, CD45, CD56, CD73, CD105, HLA-DR).Inkqubo yokufakwa kombala wesiginali yenziwa ngokumisela itshaneli eluhlaza kumfanekiso we-PE fluorescence, kunye nebala eliqaqambileyo.Ulwahlulo lwesalathiso somfanekiso webala eliqaqambileyo lasetyenziswa njengemaski ukwenza isampulu yesiginali ye-PE fluorescence.Iziphumo zeCD105 zaboniswa (Figure 5).
Umzobo 5: Iziphumo ze-CD105 ze-AdMSC zagqitywa ngu-Countstar Rigel.A: Uhlalutyo lobungakanani bepesenti elungileyo ye-CD105 kwiisampulu ezahlukeneyo yi-FCS Express 5 kunye nesoftware.B: Imifanekiso ekumgangatho ophezulu ibonelela ngolwazi olongezelelweyo lwe-morphological.C: Iziphumo eziqinisekisiweyo nge-thumbnails nganye yeseli enye, izixhobo ze-software ze-FCS zohlula iiseli zibe ngamaqela ahlukeneyo ngokokubonakaliswa kwazo kweprotheyini.
Ezinye iziphumo ze-antibodies ziboniswe kwi-Fig 6
Umzobo 6: A: Umfanekiso omele ii-ASC ezinemopholoji emile okwe spindle.Ibanjwe yi-OLYMPUS microscope.Ukwandiswa koqobo, (10x).B: Ukwahlulahlula kwe-Adipogenic yee-ASC kungqinwa yi-Ruthenium Red staining ebonisa iindawo zokumbiwa kwezimbiwa.Ibanjwe yi-OLYMPUS microscope.Ukwandiswa koqobo (10x).C: I-Countstar FL yeempawu ze-ASCs.
Isishwankathelo:
I-Countstar FL inokubeka iliso kugxininiso, ukusebenza, kunye neempawu ze-phenotype (kunye neenguqu zabo) ngexesha lokuvelisa kunye nokwahlula kwezi seli ze-stem.I-FCS Express ibonelela ngomsebenzi wokuphonononga yonke iseli yomqondiso, qinisekisa idatha ngomfanekiso.Umsebenzisi unokuba nokuzithemba ukwenza imifuniselo elandelayo esekwe kwiziphumo ze-Countstar Rigel.I-Countstar Rigel ibonelela ngendlela ekhawulezayo, ephucukileyo, kunye nethembekileyo yokulawula umgangatho weeseli zesiqu.
