CN1718727A - 干细胞亚群分离培养及治疗肝损伤疾病的制备工艺 - Google Patents
干细胞亚群分离培养及治疗肝损伤疾病的制备工艺 Download PDFInfo
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Abstract
本发明公开了一种干细胞亚群治疗肝损伤的制备工艺，涉及了首次利用一类干细胞亚群进行细胞移植及应用。从骨髓中分离、培养出一类干细胞亚群，具有多系分化的潜能。经克隆扩增后的细胞主要免疫表型为30代以上始终保持Flk1阳性。经细胞移植将Flk1+干细胞亚群植入体内受损肝脏，能够启动肝组织的内源性修复，植入的供体细胞部分分化为肝脏组织特异性细胞，修复受损肝脏。对CCl4导致的小鼠肝纤维化模型进行Flk1+干细胞亚群移植后，肝纤维化得到明显改善。这为临床治疗肝脏肿瘤、肝炎、肝衰竭、纤维化、硬化等肝损伤疾病提供了新的途径。
Description
本发明首次利用一类干细胞亚群进行细胞移植治疗肝损伤疾病。从骨髓中分离、培养的一类干细胞亚群，具有多系分化的潜能。经克隆扩增后的细胞主要免疫表型为30代以上始终保持CD34，CD45，CD31，vWF，GlyA，CD11a，CD11b阴性，Flk1阳性。通过细胞移植将Flk1+干细胞亚群植入体内受损肝脏，能够启动肝组织的内源性修复，植入的供体细胞部分分化为肝脏组织特异性细胞，修复受损组织。应用CCl4导致的小鼠肝纤维化模型进行Flk1+干细胞亚群尾静脉注射细胞移植后，肝纤维化得到明显改善。这就为临床治疗肝脏肿瘤、肝炎、纤维化、硬化等肝损伤疾病提供了新的途径。
由于各种病因引起的肝脏损伤，尤其是肝纤维化及其终末阶段的肝硬化，是威胁人类健康的一种严重疾患。目前，对于肝硬化的治疗仅限于去除潜在的有害刺激，用干扰素、病毒唑和lamivudine等对病毒性肝炎进行抗病毒治疗，以及最后的治疗手段，即肝移植。肝移植是治疗末期肝硬化的有效手段，但是由于移植器官的来源有限，需要移植的患者数量众多，以及一些因素的限制导致并非每个患者都适于进行移植，因此迫切需要发展有效的治疗手段。该种干细胞亚群具有自我更新和多分化能力，由于其易获得、扩增等诸多优点，成为细胞移植的首选种子细胞。本发明在国际上首次将干细胞亚群用于细胞移植治疗肝脏损伤疾病，并建立了细胞移植的安全有效方法，为临床治疗肝脏肿瘤、肝炎、肝衰竭、纤维化、硬化等肝损伤疾病打下了基础。
本发明的目的是通过下述方法实现的：
一.Flk1+干细胞亚群的分离和培养：
从雄性BALB/c小鼠骨髓分离Flk1+干细胞亚群，用Ficoll淋巴细胞分离液(密度1.077g/ml)分离单个核细胞，用MACS CD45，GlyA和CD34磁珠去除造血细胞。将经过分选的细胞以每孔1，5，10个细胞的密度接种于96孔板中(共15块)，用含有胎牛血清、抗坏血酸的扩增培养液在含5％的CO2，37℃培养箱内孵育。在接种后24小时内标记只有单个细胞贴壁的孔，每日观察是否有细胞克隆出现，对出现1个以上克隆的孔予以丢弃。用胰酶消化、扩增细胞克隆，这些克隆来源的细胞主要免疫表型为30代以上始终保持CD34，CD45，CD31，vWF，GlyA，CD11a，CD11b阴性，而Flk1阳性。因而我们称之为Flk1+干细胞亚群。
二.CCl4肝损伤模型建立和Flk1+干细胞亚群的输入
将雌性BALB/c鼠随机分为四组(20只/组)。1-3组均给予CCl4灌胃(1ml/公斤体重，溶解在150μl玉米油中)，每周2次，直到在接受首剂CCl4 2或5周后被处死为止。来自雄性BALB/c鼠的Flk1+干细胞亚群经尾静脉注射输入受体鼠(106/只)，其中第2组小鼠在接受CCl4后立即进行细胞注射，第3组是在首剂CCl4的一周后接受供体细胞移植。第1组仅接受等体积的生理盐水注射。第4组小鼠只给予玉米油，作为正常对照。在接受CCl4 2或5周后，处死小鼠。用4％多聚甲醛固定肝左叶，石蜡包埋，切片。肝的其他的部分则用液氮冷冻，保存在-70℃，用于提取基因组DNA和总RNA。收集血液，评价肝功能指标和肝纤维化。
三.组织学检测和免疫荧光染色
实验组肝脏切片用苏木素-伊红常规染色或行Masson三色染色来检测肝脏变性、坏死的范围或纤维化的程度。CCl4处理2周后，各实验组小鼠的肝脏均可见广范的变性/坏死，但其程度在1-3组之间并没有显著差异(图2A)。而在5周以后，第2组小鼠肝脏变性/坏死的范围则较1、3组明显缩小(图2B)。Masson三色染色法显示用CCl4处理5周可以导致显著的肝纤维化形成。第一组小鼠的肝脏显示有桥状连接形成(图3A)，而在接受首剂CCl4后就立即进行Flk1+干细胞亚群移植的受体鼠纤维化程度明显减轻(图3B)，但是在首剂CCl4后1周再接受Flk1+干细胞亚群移植者肝纤维化程度与第一组相比无显著差异(图3C)。第4组小鼠的肝脏则无纤维化发生(图3D)。
用山羊抗鼠ALB的多克隆抗体和Cy5标记的兔抗山羊二抗进行ALB的免疫荧光染色，用小鼠Y染色体特异性探针行Y染色体荧光原位杂交(FISH)检测。在CCl4损伤5周后处死的雌性受体小鼠的肝脏里，用Y染色体FISH的方法能够检测到来自雄性供体的细胞(绿色信号)，同时免疫组化证实它们具有上皮细胞表型，能够表达ALB(蓝色信号)。这种现象可见于第2组和第3组小鼠(图1A)。另外，Sry基因的PCR证实在第2和第3组雌性受体小鼠的肝脏中均有来自雄性供体的细胞(图1B)。但是，在CCl4损伤2周后处死的第2和第3组小鼠的肝脏中，我们未检测到ALB和Y-染色体双阳性的细胞(表1)。这些结果提示供体来源的Flk1+干细胞亚群在CCl4损伤的受体小鼠的肝脏中能够分化为产生ALB的细胞，但这一向组织特异性细胞分化的过程是逐渐发生的，可能需要2周以上。
四.肝脏的羟脯氨酸含量和血清学检测
CCl4损伤也可能导致肝脏内胶原沉积的增加，因此，我们比较了各组肝脏中羟脯氨酸(胶原纤维所特有的一种修饰氨基酸)的含量。检测方法与Fukui等相同，用200mg冰冻标本测定[1]。CCl4刺激5周后，第1组小鼠肝脏羟脯氨酸含量为516±84μg/g，明显高于正常对照组(48±7μg/g)。相反，在接受首剂CCl4后就立即进行Flk1+干细胞亚群移植的受体鼠肝脏羟脯氨酸含量为128±21μg/g，尽管明显高于正常对照，但与第1组相比已明显降低。在首剂CCl4后1周再接受Flk1+干细胞亚群移植的小鼠，肝脏羟脯氨酸含量有所降低(482±69μg/g)，但与第1组相比无统计学差异。
肝脏羟脯氨酸含量的谷丙转氨酶和总胆红素用我们医院的常规方法测定。血清透明质酸(HA)和III型前胶原蛋白(P-III-P)的测定方法与Yoshigi等人相同[2]。与第1组相比，第2组小鼠血清中的纤维化标志物HA、P-III-P和ALT均明显降低，而第3组则不然。1-3组血清中总胆红素水平无明显差异(表1)。
五.PCR检测
用PCR的方法分析雄性特异性Sry基因，来检测雌性受体小鼠肝脏中来自雄性供体的细胞，引物为5′-CAG CTA ACA CTG ATC TTTTC-3′和5′-TTA CTG AGC CAG AAT CAT AG-3′，从肝脏提取的基因组DNA作为扩增模板。反应条件如下：94℃孵育3分钟；35个循环(94℃30秒，50℃30秒，72℃2.5分钟)；最后在72℃延伸5分钟。
另外，由于TGF-β1是与胶原合成有关的重要纤维化因子之一，用半定量PCR的方法检测纤维化标志物转化生长因子-β1(transforming growth factor-β1，TGF-β1)和α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)的表达，用β-肌动蛋白(β-actin)作为管家基因。用Trizol提取总RNA，并用紫外分光光度计定量。以oligo-dT(18)为引物从4μg总RNA合成第一链cDNA。逆转录产物的五十分之一用于PCR扩增，总反应体系为20μL，包含10pmole引物，200μM的dNTPs，和2个单位Taq聚合酶(Gibco)。引物如下：TGF-β1(498bp)，上游，5’-AAT ACG TCA GAC ATT CGG GAAGCA-3′，下游，5′-GTC AAT GTA CAG CTG CCG CAC ACA-3′；α-SMA(368bp)，上游，5′-CTG GAG AAG AGC TAC GAA CTG C-3′，下游，5′-CTG ATC CAC ATC TGC TGG AAG G-3′；β-肌动蛋白(319bp)，上游，5′-TGT ACG TAG CCA TCC AGG C-3′，下游，5′-TTCTCC AGG GAG GAA GAG GA-3′。PCR过程包括95℃5分钟，30个循环(95℃变性30秒，60℃(TGF-β1)或62℃(α-SMA)退火30秒，72℃延伸90秒)，和最后在72℃延伸10钟。PCR产物电泳后用紫外扫描，并GelWorks软件进行定量分析。研究结果表明：第1和第3组的TGF-β1 mRNA水平明显高于第2组(图3E)。
另外，在第1和第3组中可以检测到α-SMA(一种肝星状细胞激活的标志物)的强表达，而在第2组中明显降低(图3E)。
六.统计分析
为了比较各组间的数据是否有统计学差异，用Mann-Whitney U检验来比较两组间的均值，用Kruskal-Wallis检验来比较两组以上各组间平均值的差异。当P＜0.05时认为有统计学意义。
附图说明
图1 雌性受体肝脏中的Sry基因和ALB+/Y染色体+细胞。
(A)用抗ALB抗体进行免疫荧光染色，蓝色信号标志为ALB阳性细胞；行Y染色体FISH，绿色信号标志Y染色体阳性的肝细胞。
(a)第1组ALB-/Y染色体- (b)第2组ALB+/Y染色体+
(c)第3组ALB+/Y染色体+ (d)第4组ALB-/Y染色体-
(B)PCR检测四组受体小鼠的肝脏细胞中Sry基因
图2 CCl4处理2或5周后各组小鼠肝脏常规HE染色。
(A)CCl4处理2周以后
(B)CCl4处理5周以后
图3 注射Flk1+干细胞亚群减轻四氯化碳导致的肝纤维化。
(A)对第1组小鼠的肝脏切片行Masson三色染色
(B)对第2组小鼠的肝脏切片行Masson三色染色
(C)对第3组小鼠的肝脏切片行Masson三色染色
(D)对第4组小鼠的肝脏切片行Masson三色染色
(E)用RT-PCR检测各组肝脏中TGF-β1和α-SMA的表达，并以β-actin作为管家基因。
表1 Flk1+干细胞亚群在CCl4作用2或5周(W)后对小鼠肝功能的影响
表1
| Mice | HA(ng/mL) | P-III-P(ng/mL) | ALT(U/mL) | Total bilirubin(g/L) | ALB+/Y-chromosome+cells | |||||
| 2W | 5W | 2W | 5W | 2W | 5W | 2W | 5W | 2W | 5W | |
| G1G2G3G4 | 145.2±18.429.8±11.6*134.8±19.89.6±2.0 | 181.3±23.439.1±51*183.1±27.6+9.8±2.1 | 29.8±4.08.9±2.7*30.1±4.11.8±0.7 | 39.7±5.213.1±2.9*37.9±4.91.9±07 | 189.2±37.2136.8±28.7*181.8±24.941.9±7.1 | 216.3±47.679.6±6.4*213.7±37.637.7±6.9 | 120.1±20.5117.4±23.1122.6±22.550.4±20.7 | 124.8±21.2121.4±20.1120.2±23.151.1±20.4 | ---- | -++- |
Data are means±SD(n＝10).
*p＜0.01与G1组小鼠相比；p＞0.05与G1组小鼠相比。
引证文献：
1.Yoshiji H，Kuriyama S，Miyamoto Y，et al.Tissue inhibitor ofmetalloproteinases-1 promotes liver fibrosis development in atransgenic mouse model.Hepatology 2000；32：1248.
2.Yoshiji H，Kuriyama S，Yoshii J，et al.Angiotensin-II type 1 receptorinteraction is a major regulator for liver fibrosis development in rats.Hepatology 2001；34：745.
本发明的优点：
1.本发明首次报道了利用一类干细胞亚群进行细胞移植治疗肝脏损伤疾病的过程，并证明了干细胞亚群分化为肝脏组织特异性细胞和具有潜在的植入肝组织的能力；
2.本发明建立了一类干细胞亚群进行细胞移植的有效方法，操作安全可行；
3.本发明为治疗肝脏肿瘤、肝炎、肝衰竭、纤维化、硬化等肝损伤疾病提供了实验和临床研究基础，为细胞移植治疗提供了新途径。
Claims (4)
1.一种干细胞亚群治疗肝损伤的制备工艺，涉及利用一类干细胞亚群进行细胞移植及应用。其特征在于：从骨髓中分离、培养的一类干细胞亚群，具有多系分化的潜能。经克隆扩增后的细胞主要免疫表型为30代以上始终保持Flk1阳性。通过细胞移植将Flk1+干细胞亚群植入体内受损肝脏，能够启动肝组织的内源性修复，植入的供体细胞部分分化为肝脏组织特异性细胞，修复受损组织。应用CCl4导致的小鼠肝纤维化模型进行Flk1+干细胞亚群移植后，肝纤维化得到明显改善。这就为临床治疗肝脏肿瘤、肝炎、肝衰竭、纤维化、硬化等肝损伤疾病提供了新的途径。
2.根据权利要求1所述的一类干细胞亚群的制备工艺，其特征在于：从骨髓中分离出的干细胞亚群具有多系分化潜能，经克隆扩增30代以上始终保持干细胞主要免疫表型。如CD34，CD45，CD31，vWF，GlyA，CD11a，CD11b阴性，Flk1阳性等。
3.根据权利要求1所述的一类干细胞亚群应用于体内治疗肝损伤疾病的制备工艺，其特征在于：通过细胞移植将Flk1+干细胞亚群植入体内受损肝脏，能够启动肝组织的内源性修复，植入的供体细胞可分化为肝脏组织特异性细胞。如细胞移植利用静脉注射的方法，Flk1+干细胞亚群经血运到达并停泊在肝脏受损部位；植入的供体细胞部分显现为上皮细胞形态，并表达白蛋白等。
4.根据权利要求1所述的一类干细胞亚群应用于体内治疗肝脏肿瘤、肝炎、肝衰竭、肝纤维化、肝硬化的制备工艺，其特征在于：从骨髓中分离培养出的这一类干细胞亚群通过细胞移植应用于临床，治疗肝脏肿瘤、肝炎、肝衰竭、肝纤维化及其终末阶段的肝硬化等等各种病因引起的肝脏损伤疾病。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409366A (zh) * | 2013-02-20 | 2013-11-27 | 潘骏 | 成人多能祖细胞的培养基以及采用成人多能祖细胞的培养基培养成人多能祖细胞的方法 |
| RU2822653C1 (ru) * | 2023-08-18 | 2024-07-11 | Государственное бюджетное учреждение здравоохранения города Москвы городская клиническая больница имени С.П. Боткина департамента здравоохранения города Москвы (ГБУЗ ГКБ им. С.П. Боткина ДЗМ) | Способ регенеративной клеточной терапии для лечения гепатоцеллюлярной недостаточности при циррозе печени |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409366A (zh) * | 2013-02-20 | 2013-11-27 | 潘骏 | 成人多能祖细胞的培养基以及采用成人多能祖细胞的培养基培养成人多能祖细胞的方法 |
| RU2822653C1 (ru) * | 2023-08-18 | 2024-07-11 | Государственное бюджетное учреждение здравоохранения города Москвы городская клиническая больница имени С.П. Боткина департамента здравоохранения города Москвы (ГБУЗ ГКБ им. С.П. Боткина ДЗМ) | Способ регенеративной клеточной терапии для лечения гепатоцеллюлярной недостаточности при циррозе печени |
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