Abstract: Amaseli we-Mesenchymal stem ayisethi engaphansi yamaseli e-pluripotent stem angahlukaniswa ne-mesoderm.Ngezimpawu zabo zokuvuselela ukuziphindaphinda kanye nezici zokuhlukanisa izinkomba eziningi, banamandla amakhulu ezindlela zokwelapha ezihlukahlukene kwezokwelapha.Amaseli we-Mesenchymal stem ane-phenotype eyingqayizivele ye-immune kanye nekhono lokulawula amasosha omzimba.Ngakho-ke, amangqamuzana e-mesenchymal stem asevele asetshenziswa kabanzi ekufakweni kwe-stem cell, ubunjiniyela bezicubu, kanye nokufakelwa kwezitho zomzimba.Futhi Ngale kwalezi zinhlelo zokusebenza, asetshenziswa njengethuluzi elikahle kubunjiniyela bezicubu njengamaseli embewu ochungechungeni locwaningo oluyisisekelo nolwezempilo.Kuze kube manje, ayikho indlela eyamukelwa kabanzi kanye nendinganiso yokulawula ikhwalithi yamaseli e-mesenchymal stem.I-Countstar Rigel ingaqapha ukugxila, ukusebenza, kanye nezici ze-phenotype (kanye nezinguquko zazo) phakathi nokukhiqizwa nokuhlukaniswa kwalawa maseli esiqu.I-Countstar Rigel futhi inenzuzo ekutholeni ulwazi olwengeziwe lwe-morphological, olunikezwa indawo ekhanyayo ehlala njalo kanye nokuqoshwa kwesithombe okusekelwe ku-fluorescence phakathi nayo yonke inqubo yokuqapha ikhwalithi yeseli.I-Countstar Rigel inikezela ngendlela esheshayo, eyinkimbinkimbi, nethembekile yokulawula ikhwalithi yama-stem cells.
Izimpahla nezindlela:
Amaseli esiqu se-mesenchymal (AdMSC) asuselwa ku-adipose aphiwe uProfesa Nianmin Qi, isixazululo se-AO/PI (i-Shanghai RuiYu, CF002).I-antibody: CD29, CD34, CD45, CD56, CD73, CD105, HLADR (BD Company).
Ama-AdMSC akhuliswe ku-37℃, 5% CO2 incubator humidified.Gaya nge-trypsin ngaphambi kokusetshenziswa.
Inqubo yokungcolisa umaka we-CD yalandelwa njengemanuwali yama-antibody.
Ukutholwa komaka we-CD nge-Countstar Rigel:
1. Inqubo yokufaka umbala wesignali idalwe ngokusetha isiteshi se-PE esithombeni se-PE fluorescence.
2. Izinkambu ezi-3 zathathwa ekamelweni ngalinye.
3. Ngemuva kokuthi ukuthwebula isithombe kanye nokuhlaziywa kokuqala sekuqediwe, ukumiswa kwe-threshold (log gate) ukuze kudluliselwe okuhle nokubi kwamiswa isofthiwe ye-FCS.
Ukulawulwa kwekhwalithi ye-stem cell
Umfanekiso olandelayo (Umdwebo 1) ubonisa inqubo ye i-stem cell therapy .
Umfanekiso 1: Inqubo yokwelashwa kwe-stem cell
Imiphumela:
Ukunquma ukugxila, ukusebenza, ububanzi, nokuhlanganisa ama-AdMSC.
Ukusebenza kwama-AdMSC kwanqunywa i-AO/PI, Inqubo yokufaka imibala emibili yadalwa ngokumisa isiteshi Esikuhlaza kanye neshaneli Ebomvu ukuze kufane ne-AO ne-PI fluorescence, kanye nenkambu ekhanyayo.Izithombe eziyisibonelo zibonisiwe kuMfanekiso 2.
Umfanekiso 2. Izithombe zangaphambi kokuthutha nangemuva kokuthuthwa kwama-AdMSC.A. Ngaphambi kokuthutha;isithombe esimele siyaboniswa.B. Ngemva kokuhamba;isithombe esimele siyaboniswa.
Ukusebenza kwama-AdMSC kwashintshwa kakhulu ngemva kokuhamba uma kuqhathaniswa nangaphambili kwezokuthutha.Ukusebenza ngaphambi kokuthutha bekungama-92%, kodwa kwehle kwafika ku-71% ngemuva kokuhamba.Umphumela uboniswe kuMfanekiso 3.
Umfanekiso 3. Imiphumela yokusebenza kwama-AdMSC (Ngaphambi kokuthutha nangemuva kokuthutha)
Ububanzi nokuhlanganisa kuphinde kwanqunywa ngu-Countstar Rigel.Ububanzi be-AdMSC bushintshwe kakhulu ngemva kokuhamba uma buqhathaniswa nangaphambili kwezokuthutha.Ububanzi ngaphambi kokuthutha babungu-19µm, kodwa bunyuke baba ngu-21µm ngemva kokuthutha.Ukuhlanganiswa kwezinto zangaphambili zokuthutha bekungama-20%, kodwa kukhuphuke kwafinyelela ku-25% ngemuva kokuhamba.Kusukela ezithombeni ezithwetshulwe ngu-Countstar Rigel, i-phenotype ye-AdMSCs yashintshwa kakhulu ngemva kokuhamba.Imiphumela ikhonjiswe kuMfanekiso 4.
Umfanekiso 4: Ububanzi kanye nemiphumela yokuhlanganisa.IMP: Izithombe ezimele ama-AdMSC, uhlobo lwe-AdMSC lushintshwe kakhulu ngemva kokuthutha.B: Ukuhlanganisa ngaphambi kokuthutha bekungama-20%, kodwa kukhuphuke kwafinyelela ku-25% ngemuva kokuhamba.C: Ububanzi ngaphambi kokuthutha babungu-19µm, kodwa bunyuke baba ngu-21µm ngemva kokuthutha.
Thola i-immunophenotype ye-AdMSCs ka-Countstar Rigel
I-immunophenotype ye-AdMSCs yanqunywa i-Countstar Rigel, i-AdMSCs yafakwa amasosha omzimba ahlukene ngokulandelana (CD29, CD34, CD45, CD56, CD73, CD105, HLA-DR).Inqubo yokufaka umbala wesignali idalwe ngokusetha isiteshi Esikuhlaza ukuze senze isithombe se-PE fluorescence, kanye nenkambu ekhanyayo.Isegimenti yereferensi yenkundla ekhanyayo isetshenziswe njengemaski ukuze kusampula isignali ye-PE fluorescence.Imiphumela ye-CD105 ikhonjisiwe (Umfanekiso 5).
Umfanekiso 5: Imiphumela ye-CD105 ye-AdMSC inqunywe ngu-Countstar Rigel.A: Ukuhlaziywa komthamo wephesenti elihle le-CD105 kumasampuli ahlukene nge-software ye-FCS express 5 plus.B: Izithombe zekhwalithi ephezulu zinikeza ulwazi olwengeziwe lwe-morphological.C: Imiphumela eqinisekisiwe ngezithonjana zawo wonke amaseli, amathuluzi esofthiwe ye-FCS ahlukanisa amaseli ngamaqembu ahlukene ngokuya ngokwehlukana kwawo kwe-protein expression.
Eminye imiphumela yamasosha omzimba ikhonjisiwe ku-Fig 6
Umfanekiso 6: A: Umfanekiso omele ama-ASC ane-morphology emise okwe-spindle.Kuthwetshulwe ngesibonakhulu i-OLYMPUS.Ukukhulisa koqobo, (10x).B: Ukuhlukaniswa kwe-Adipogenic kwama-ASC kufakazelwa yi-Ruthenium Red staining ekhombisa izindawo zokumbiwa phansi.Kuthwetshulwe ngesibonakhulu i-OLYMPUS.Ukukhulisa koqobo (10x).C: Ukulingiswa kwe-Countstar FL kwama-ASC.
Isifinyezo:
I-Countstar FL ingaqapha ukugxila, ukusebenza, kanye nezici ze-phenotype (kanye nezinguquko zazo) phakathi nokukhiqizwa nokuhlukaniswa kwalawa maseli esiqu.I-FCS Express inikeza umsebenzi wokubuyekeza wonke amaseli wesignali, uqinisekise idatha ngesithombe.Umsebenzisi angaphinda abe nesibindi sokwenza izivivinyo ezilandelayo ngokusekelwe emiphumeleni ye-Countstar Rigel.I-Countstar Rigel inikezela ngendlela esheshayo, eyinkimbinkimbi, nethembekile yokulawula ikhwalithi yama-stem cells.
