干細(xì)胞研討會(huì)課件

1、干細(xì)胞治療不同病因所致終末期肝病的研究進(jìn)展終末期肝病肝炎病毒乙肝 9300萬丙肝 5000萬 J Hepatol, 2010終末期肝病危害嚴(yán)重每年30萬人死于終末期肝??？終末期肝病治療困難2500 人/年成體干細(xì)胞具有可塑性干細(xì)胞肝細(xì)胞終末期肝病?23415病毒感染性肝病11酒精性肝病非酒精性肝病遺傳和代謝性肝病免疫性肝病自身免疫性肝炎原發(fā)性膽汁性肝硬化自身免疫性肝病原發(fā)性硬化性膽管炎AIHPBCPSCJ Hepatol. 2003 Nov;39(5):679-85干細(xì)胞與自身免疫性肝病13 AIH-1 patients, 13 PBC patients, 12 cirrhotic contr

2、ols (CC) 10 healthy controls (HC).造血祖細(xì)胞在AIH-1患者中增多，在PBC患者中缺失AIH-1（ P0.05） PBC （ P0.05） No differences between CC and HCCD34+, CD34+/CD38+ CD34+/CD38- cells具有功能的祖細(xì)胞粒巨噬細(xì)胞集落生成單位 瀑式紅細(xì)胞集落形成單位 Arch Pharm Res. 2012 Feb;35(2):213-21干細(xì)胞與自身免疫性肝病無論是否伴有肝硬化或服用免疫抑制劑，AIH-1 和 PBC多伴有骨髓單核細(xì)胞增多淋巴細(xì)胞增多幼稚巨核細(xì)胞增多AIH-1、PBC患者

3、骨髓異常13 例AIH-1 13 例PBC 12 例非免疫性肝硬化8 例健康對照 干細(xì)胞與自身免疫性肝病J Autoimmun. 2005 Dec;25(4):283-8. PBC 和AIH-1患者的細(xì)胞因子微環(huán)境明顯不同于正常和非免疫性肝病患者，且PBC與AIH-1亦不同IL-4, TNF-alpha TGF-betaBM IL-10IL-10, IL-4 TNF-alphaBM IFN-gamma13 例AIH-1 13 例PBC 7 例乙肝肝硬化10 例健康對照 J Hepatol. 2005 Mar;42(3):393-9干細(xì)胞與自身免疫性肝病AIH-1和PBC患者的骨髓單個(gè)核細(xì)胞的凋

4、亡、巨噬細(xì)胞活化以及血細(xì)胞生成的抑制性細(xì)胞因子均高于對照組。凋亡標(biāo)記分子和CD14增高(P0.0001). Fas+ 細(xì)胞與凋亡細(xì)胞成正相關(guān)d (P=0.0001)TNF-alpha and IFN-gamma 在AIH-1 (P=0.003 and P=0.001) 和 PBC (P=0.0001)中增高兩組對照間無差異13 例AIH-1 13 例PBC 10 例健康對照 12例肝硬化1997non-Hodgkins lymphoma2003allogenic-PBSCT 2004LKM1-positive AIH-2Am J Gastroenterol. 2008 May;103(5):1

5、313-4.干細(xì)胞與自身免疫性肝病異體干細(xì)胞移植可以誘發(fā)自身免疫性肝病的發(fā)生。肝組織出現(xiàn)碎片樣壞死，單核細(xì)胞浸潤.與移植前相比，肝功異常一個(gè)月（lane3）和10個(gè)月時(shí)患者血清中出現(xiàn)抗體GFP(+/-)骨髓細(xì)胞 C57BL/6 recipients移植4周后膽管結(jié)扎J Gastroenterol Hepatol. 2007 Nov;22(11):2001-8干細(xì)胞與自身免疫性肝病骨髓細(xì)胞參與了膽管結(jié)扎時(shí)導(dǎo)管周圍纖維化的發(fā)生。第7天時(shí)，導(dǎo)管周圍纖維化區(qū)域包含成纖維細(xì)胞特異性蛋白陽性細(xì)胞。且部分細(xì)胞為GFP+細(xì)胞血清轉(zhuǎn)氨酶自身免疫性抗體膽管周圍單核細(xì)胞浸潤外周血和淋巴結(jié)中調(diào)節(jié)性T細(xì)胞增多TGF-1

6、 增高IFN- 下降 IL-10不變 干細(xì)胞與自身免疫性肝病Clin Exp Med. 2011 Mar;11(1):25-32 injecting C57BL/6 mice with polyI:CBM-MSC降低PBC小鼠機(jī)體免疫反應(yīng)，促進(jìn)肝臟炎癥恢復(fù)MSC infusionAfter 6 weeks Hepatology. 2010 Jan;51(1):267-76干細(xì)胞與自身免疫性肝病骨髓細(xì)胞移植可以改善硬化性膽管炎小鼠的肝功能Abcb4(-/-)移植與Abcb4(+/+) 無差異H&E, Masson Trichrome sham-Tx controlsyngeneic BM-Tx

7、allogeneic BM-Tx23415病毒感染性肝病11酒精性肝病非酒精性肝病遺傳和代謝性肝病免疫性肝病遺傳和代謝性肝病糖原貯積病血色病WD酪氨酸血癥。BMC Gastroenterol. 2011正常骨髓細(xì)胞移植治療WD小鼠：早期階段優(yōu)于晚期階段（改善肝功能和銅代謝）late stage (5 months of age)early stage(2 months of age)WD mice正常骨髓細(xì)胞 生理鹽水WD 骨髓細(xì)胞 1, 4, 8 and 12 weekscopper concentrationAST donor cell populationInt J Biochem Ce

8、ll Biol. 2012 Apr;44(4):648-58. 小鼠胚胎干細(xì)胞來源的肝細(xì)胞可以糾正肝臟的代謝紊亂ES cell-derived hepatocytes Fah(-/-) mice可見肝臟再生FAH+肝細(xì)胞可以糾正代謝紊亂、降低死亡23415病毒感染性肝病11酒精性肝病非酒精性肝病遺傳和代謝性肝病免疫性肝病非酒精性肝病患者外周循環(huán)中內(nèi)皮祖細(xì)胞數(shù)量減少，心腦血管疾病風(fēng)險(xiǎn)增高PLoS One. 2012;7(2):e31799. Epub 2012 Feb 16干細(xì)胞與非酒精性肝病代謝綜合征；心肌梗死；高尿酸血癥；腰圍；體重；血糖；甘油三脂循環(huán)中 EPCEPC的功能NAFLD 可以作

9、為預(yù)后判定因素J Hepatol. 2011 Nov;55(5):1112-20MSC通過阻抑炎癥反應(yīng)而非逆轉(zhuǎn)代謝反應(yīng)發(fā)揮肝保護(hù)作用干細(xì)胞與非酒精性肝病血清肝酶增高、肝腫大、肝纖維化、炎細(xì)胞浸潤、甘油三酯沉積、纖維化標(biāo)志分子表達(dá)增高、致炎因子增高只有脂肪變性23415病毒感染性肝病11酒精性肝病非酒精性肝病遺傳和代謝性肝病免疫性肝病Liver biopsies AH 患者肝組織中以及外周血中CD34(+) 細(xì)胞增多(P 0.05) 7.9%-26.8%的肝纖維母細(xì)胞為骨髓來源骨髓細(xì)胞對肝臟實(shí)質(zhì)細(xì)胞無影響Stem Cells Dev. 2010 Sep;19(9):1417-25酒精性肝損傷可以

10、動(dòng)員CD34+細(xì)胞進(jìn)入外周循環(huán)而定植于肝臟， 通過作用于纖維母細(xì)胞群而非肝臟實(shí)質(zhì)細(xì)胞促進(jìn)肝細(xì)胞修復(fù)接受女性肝移植的男性酒精性肝病患者Transplant Proc. 2005 Jul-Aug;37(6):2563-6急性肝切除或肝硬化急性失代償期不能動(dòng)員骨髓細(xì)胞Hepatology. 2008 Jul;48(1):221-9.G-CSF 注射后7天可以動(dòng)員酒精性肝炎患者 CD34+ cells, 促進(jìn)HGF分泌, 誘導(dǎo) HPC 增殖A G-CSF (10 mg /kg/day), 5天；n=13CD34+ 細(xì)胞增多、HGF 增高、Ki67+/CK7+ 細(xì)胞與 CD34+ 細(xì)胞成正相關(guān)B內(nèi)科治療

11、n=11兩組的中性粒細(xì)胞與組織學(xué)改變相似 Stem Cells Dev. 2011 Sep;20(9):1503-10. 自體骨髓細(xì)胞移植可以提高酒精性肝硬化患者的肝功能 5例酒肝患者、 5 例酒肝對照自體骨髓細(xì)胞移植隨訪24 周隨訪期間，白蛋白、總蛋白、凝血酶原時(shí)間、顯著改善3例Child 評分B級(jí)的患者下降； 3例患者骨髓活化Am J Gastroenterol. 2008 Aug;103(8):1952-8自體CD34+細(xì)胞經(jīng)體外擴(kuò)增后移植治療酒精性肝硬化安全有效動(dòng)員、分離、體外擴(kuò)增CD34+ 細(xì)胞擴(kuò)增5倍 膽紅素在移植后4, 8, 12 周下降 (P 0.05). ALT、AST改善，

12、在移植后1周最顯著（P 0.05）. 7例患者Child 評分改善5例患者腹水癥狀緩解9例酒精性肝硬化患者Clin Transplant. 2011 Jan-Feb;25(1):54-60ALT and albumin 門靜脈直徑、肝臟回聲組織學(xué)肝硬化小鼠來源的骨髓細(xì)胞并不能改善肝硬化小鼠的肝功能或肝纖維化酒精性肝硬化小鼠倍 酒精性肝硬化小鼠骨髓細(xì)胞均無改善Hepatology. 2012 Jun;55(6):1931-41肝祖細(xì)胞標(biāo)志分子與肝臟疾病的嚴(yán)重程度以及短期死亡率成正相關(guān)23415病毒感染性肝病11酒精性肝病非酒精性肝病遺傳和代謝性肝病免疫性肝病WHO and CDC fact sh

13、eets, available at and 全球60億人口20億人曾感染HBV，占全球人口1/3慢性HBV感染者3.85億，占全球人口6%，其中亞洲占2/3 ，中國占1/3 25%-40%最終將死于肝硬化或肝癌Kew MC. Viral hepatitis & liver disease, 2004每年因乙肝相關(guān)死亡75萬例！干細(xì)胞與病毒感染性肝病J Gastroenterol Hepatol, 2003干細(xì)胞移植能顯著改善肝損害大鼠的肝功能外周血干細(xì)胞在體內(nèi)外均可被誘導(dǎo)為肝細(xì)胞樣細(xì)胞Am J Hematol, 2007Biochem Biophys Res Commun, 2008創(chuàng)建了自

14、體外周血干細(xì)胞移植治療終末期肝病新技術(shù)術(shù)前13691218(月)0.50.25術(shù)前13691218(月)0.50.25術(shù)前13691218(月)0.50.25術(shù)前13691218(月)0.50.25Am J Hematol, 2007臨床探索研究干細(xì)胞移植可有效改善患者肝功能隨機(jī)對照研究Cytotherapy, 2008試驗(yàn)流程圖 干細(xì)胞移植優(yōu)于對照組病例對照研究干細(xì)胞移植延長患者的生存期病例對照研究干細(xì)胞移植提高患者生存率術(shù)后時(shí)間（年）生存率（%）機(jī)制研究促進(jìn)肝細(xì)胞增生及細(xì)胞外基質(zhì)降解Cytotherapy, 2010Sirius RedCCl4 VehicleBMSCsIL-17BMSCs

15、 + IL-17IgG2aAnti-IL-17ABCJ Hepatol, 2012干細(xì)胞移植可能通過下調(diào)IL-17改善肝功能(A 和 B)干細(xì)胞移植后不同時(shí)間點(diǎn)觀察小鼠肺臟、肝臟、脾及腎中GFP熒光強(qiáng)度。(C)PCR技術(shù)檢測接受移植組小鼠體內(nèi)供體來源的Y染色體序列濃度。(D 和 E)肝臟冰凍切片在熒光顯微鏡下觀察GFP+細(xì)胞比例（i, MSCs組; ii, HSCs組; iii,MSCs+HSCs組）干細(xì)胞移植的體內(nèi)示蹤 A：各組接受干細(xì)胞移植治療后小鼠生存率B和C：各組小鼠肝組織分別進(jìn)行H-E及天狼星紅染色D：各組纖維化面積統(tǒng)計(jì)情況E：Real-TimePCR分析I型膠原F,G 和 H：肝功

16、能測定。i, 正常對照組ii, CCl4 組 iii, MSCs 組 iv, HSCs 組v, MSCs+HSCs組MSC和HSC移植療效比較干細(xì)胞與肝臟疾病臨床研究分布圖/179 項(xiàng)3項(xiàng)來自本課題組2012-11-22CountryNumber of patientsRandomized trail,Baseline measuresSource of stem cellType of stem cell infusedNumber of infused cellsDelivery of stem cellsImproved with infusion?Follow-up period, m

17、onthJournal, yearGermany6 (3 control)NoDaily growth rates of hepatic segmentsBone marrow Sorted CD133+ cells2.412.3 106 infusedPortal vein infusionYesN/AStem Cells -2005Italy8NoCP/MELDG-CSF mobilization onlyN/AN/AYes8J Hepatol -2006Japan9NoCPBone marrow Unsorted MNCs 5.2 109 Portal vein infusionYes6

18、Stem Cells -2006England5NoAlbumin /bilirubinG-CSF mobilized peripheral bloodSorted CD34+ cells after adherence1 1062 108Hepatic artery (n = 2) or portal vein (n = 3)Yes2Stem Cells -2006Greece2NoCP/MELDG-CSF mobilized peripheral bloodUnsorted MNCs containing CD34+ cells46.93 106 CD34+/kg Peripheral v

19、ein infusionYes30Exp Hematol-2006Iran4NoMELDBone marrow Sorted CD34+ cells5.25 106 Hepatic artery infusionNo6World J Gastroenterol-2007Iran4NoMELDBone marrow MSCs31.7 106Peripheral vein infusionYes12Arch Iran Med-2007Brazil10NoAlbumin/bilirubinBone marrow Unsorted MNCs100 106 Hepatic artery infusion

20、Yes4World J Gastroenterol-2007Germany13 (7control)NoDaily growth rates of hepatic segmentsBone marrow Sorted CD133+ cells2.412.3 106 infusedPortal vein infusionYesN/ARadiology -2007 Russian47NoLiver function CP biopsy ultrasoundBone marrowbone marrow mononuclear cells 0.5-4.510 9Peripheral vein infu

21、sionYes12B Exp Biol Med-2007China2NoAlbumin/ CPG-CSF mobilized peripheral bloodUnsorted PBSCs 107108/kg.106 infusedHepatic artery infusionYes18Am J Gastroenterol-2007Italy18NoCD34+and CD133+cell number; liver functionG-CSF mobilization onlyN/AN/ANoN/AAliment Pharmacol Ther-2008China40(20 control)Yes

22、Albumin/ CPG-CSF mobilized peripheral bloodUnsorted PBSCsPBSCs 107108/kg.106 infusedHepatic artery infusionYes6Cytotherapy-2008CountryNumber of patientsRandomized trail,Baseline measuresSource of stem cellType of stem cell infusedNumber of infused cellsDelivery of stem cellsImproved with infusion?Fo

23、llow-up period, monthJournal, yearSwitzerland13YesCP/HGF/biopsyG-CSF mobilization onlyN/AN/AYes7daysHepatology-2008England9NoBilirubin/CP/ascitesG-CSF mobilized peripheral bloodSorted CD34+ cells after adherence expanded in vitro2.3 108 Hepatic artery infusionYes3Am J Gastroenterol-2008Swede8NoEnd-S

24、tageLiver Disease scoreBone marrow stem cellsUnsorted MSCs3-5107hepatic artery or portal veinYes6Eur J Gastroenterol Hepatol. -2009Austria30NoLiver function Bone marrowUnsorted MNCs containing CD34+ cells3.78 108Hepatic artery infusionYes12Eur J Gastroenterol Hepatol. -2010Egypt48NoLiver function an

25、d clinical featuresG-CSF mobilied Bone marrow stem cellsSorted CD34+ cells, amplified and partially differentiated in culture1109hepatic artery or portal veinYes12Cell transplantation-2010Egypt140 (50 control)YesLiver function and clinical featuresG-CSF mobilied Bone marrow stem cellsCD34+ and CD133

26、+0.5108hepatic artery or portal veinYes6World J Gastroenterol-2010Egypt20 (10 control)YesLiver function， Child-Pugh score,TBIL, PTautologous bone marrow mononuclear cells3.13108Yes3J Gastrointest Canc-2010Brazil8NoLiver function CP MELDBone marrowbone marrow mononuclear cells2.0-15.0108Hepatic arter

27、y infusionYes12 Liver Int-2011Iran6NoMELDBone marrow MNCs or CD133+1.310.14 109 ；6.43.2 106portal veinNo20Arch Iran Med-2011China158 (105 control)YesALB, TBIL, PT 、MELD scoreHCC , mortalityBone marrowMSC3.4 - 3.8 108Hepatic artery infusionShort- Yes Long-No 48Hepatology-2011Egypt40 (20 control)Noliv

28、er function,Child-Pugh score, MELD score, fatigue impact scale.autologous bone marrowMSC5106interhepatic or intersplenicYes6Eur J Gastroenterol Hepatol. -2011Egypt25 (10 control)NoALB、PT、TBIL、MELDautologous bone marrowMSC（differentiated or undifferentiated1106/kgintravenouslyYes6Stem Cell Rev and Re

29、p-2011China45 ( 15 control)NoALB、TBIL、MELDumbilical cordMSCs0.5106/kgintravenously（once per month，three times)Yes12J Gastroenterol Hepatol-2011干細(xì)胞來源及種類：Bone marrow（15） G-CSF mobilized peripheral blood（7+3）umbilical cord（1）Sorted CD133+ cells（2）Unsorted MNCs（7） Sorted CD34+ cells（1）MSCs（4）MSC（differentiated ）（1）MSCsSorted CD34+ cells after adherenceU