摘要
肝脏由胚胎内胚层演变而来，胚胎期肝具有重要的造血功能。人胚6周，造血干细胞(hematopoietic stem cell，HSC)从卵黄囊迁入肝，即进入了肝脏造血期。胎儿15～24周，是肝造血的旺盛期。胎肝造血以红细胞系占绝对优势，也有粒细胞系不同发育阶段的细胞，还有淋巴细胞和巨核细胞。红细胞系中以中幼红细胞的数量最多，粒细胞系数量少，出现也晚。至胚胎6月肝脏造血逐渐减弱，到出生时由骨髓造血所取代。
胃肠道内分泌细胞分泌的血管活性多肽(Vasoactive Intestinal peptide，VIP)具有多种生物学功能，当其随门静脉血流进入肝脏后，与肝细胞上的相应受体结合，内在化后被溶酶体降解，很快被清除。因此，肝脏是灭活VIP的主要场所。肝脏局部的VIP及受体对肝脏造血及造血器官的迁移起了何种作用?对造血干细胞在肝脏的横向分化有何影响也令人感兴趣。为回答以上问题，我们探讨了VIP对造血干细胞生物学行为的影响及机制。
目的
1．探讨VIP对人HSC体外增殖及集落形成的影响，以及可能机制；
2．VIP对人HSC向造血系细胞分化的影响；
3．VIP对人HSC向肝系分化的影响及可能机制，解释VIP对HSC横向分化的可能影响；
4．通过胎肝发育中VIP及VIPR含量的变化，初探胎肝造血转移机制；
5．VIP是否通过HSC上的相应受体介导发挥上述作用，HSC上VIP受体为何种亚型?
重庆医科大学博士学位论文
方法
1.采集人脐血标本，淋巴细胞分离液分离出单个核细胞;
2.免疫磁分选技术纯化人脐血CD34十细胞，流式细胞术鉴定纯度;
3.体外集落形成实验检测V正对人HSC集落形成能力的影响;
4.扩增培养计数VIP对CD34+细胞增殖影响;
5.酶联免疫化学法测定培养HSC细胞及上清TNF-a、TGF-B水平，以及A万P水
平;
6.流式细胞术检测CD34+细胞培养过程中各系分化细胞比例;
7.免疫组织化学法检测 HSC上肝系标志AFP、ALB、CK-19的表达;
8·Westem blot方法检测Hse上ALB表达;
9.巢式RpPCR方法检测HSC上AFP、ALB的mRNA表达，随机选送ALB产
物测序;
10.采集发育不同期SD大鼠肝脏标本;
11.生物分子相互作用系统(Biacore)测定人HSC、肝脏组织vIPR结合力;
12.放射免疫法检测肝脏组织VIP浓度水平;
13.Rl’- PCR方法检测人HSC、肝脏组织VIPR的mRNA表达。
结果
1.vIP在10一7m。呱一10一‘2 mof几浓度范围内可抑制造血干细胞集落形成(抑制率
)26.97士13.72%)，P<0.05;在10一8 mol/L抑制作用最大。10·8 moULv护作用
下HSC扩增倍数第7、10、14天分别依次为1 1 .78士4.39、16.71士2.98、21.69
士3.28倍，显著低于对照组(20.13士3.32、25.64士3.51、25.33士2.61)，P<0.05。
2.vIP作用于HSC后，细胞内TNF一a浓度为149.15p创ml，较对照组显著升高，
P<0.05，增加T 49%;V正显著增加T HSC内TGF一B，浓度，为116.lop岁ml，
较对照组升高44.41%，P<0·05。
重庆医科大学博士学位论文
3.1护mo呱的v正作用Hse 14天后，粒系eD13细胞比例为50.33%，对照组
为93.35%，有显著降低，P<0.05;7天组、14天组单核系CD14细胞比例分别
依次为8.20%和15.巧%，对照组分别依次为13.80%和22.55%，也有显著降低，
P<0.05。红系CD71+细胞、巨核系CD41+细胞、B淋巴细胞CD19+细胞、T淋
巴细胞CD4/CDS细胞、祖细胞CD33+细胞在VIP作用前后均无显著性差异，
p>0.05。v正作用于CD34细胞7天后，CD34+细胞比例较对照组(18.乃%)明显
增加(22.0%)，P<0.050
4.免疫组化结果显示人HSC上不表达CK一19蛋白，但有ArP和ALB蛋白表达;
VIP作用HSC 14天后，HSC内的AFp浓度(165士8.51 Pg/ml)较对照组(270土
11.37p留ml)显著下降，P<0.05。western blot显示v正作用后Hse内ALB蛋
白表达有减弱趋势。人脐血单个核细胞和CD34+细胞都表达了AFP mRNA和
A工B mRNA，随机选取ALB产物测序与Genebank中ALB基因序列完全相同。
5.从胚胎到新生鼠，肝脏vIP量(分别为1349.18士220.37n岁加l，2439.64士394.22
ng/ml)与V正受体表达量(分别为806.67士58.47 RU/ug蛋白，952.5士121.45
RU/ug蛋白)均呈增加趋势;出生后，从未成年期到成年期肝脏vIP量(分别为
2689·47士227·53ng/ml，1911.79士453.15 ng/ml)与V护受体表达量(分别为
762.5士97.15 RU加g蛋白，425士119.38 RU/ug蛋白)都逐渐降低，即出生前后
呈相反的变化趋势。值得注意的是，新生期VIP量低于未成年期，但VIP受
体表达量却正相反。Rl’- PCR显示大鼠发育不同时期肝脏都表达VIP受体1型。
6.生物分子相互作用系统显示人造血干细胞表达 vIPR，结合量为2366.67 RU枷g
蛋白;Rl’- PCR显示人脐血造血干细胞和单个核细胞表达V正R一1 mRNA。
结论
1.VIP可抑制人造血干细胞增殖和集落形成，是一种新被认识的造血抑制剂。
2.V砰抑制人造血干细胞增殖的机制之一是通过上调HSC内造血抑制因子
TNF一a、TGF一B水平实现的。
重庆医科大学博士学位论文
3.VIP抑制造血干细胞向粒单系分化，对其向红系及其它系分化无影响;VIP从
时间上减缓CD34抗原消失，
Liver origins from endoderm and is an important hematopoietic organ in embryo. Hematopoietic stem cells (HSC) migrate to liver from yolk sac in the 6th week of embryo when liver begins its hematopoiesis and up to bloom stage during 15-24 weeks' embryo. Although granulocytes, monocytes, lymphocyte and megakaryocytes in various development stages can be found in fetal liver hematopoiesis, erythrocytes increase and mature in advance. The amount of erythroblast is especially dominant in the system of erythrocyte. Fetal liver hematopoiesis began weaken at 6 months' embryo and is replaced by bone morrow after birth.
Vasoactive intestinal peptide (VIP) secreted from endocrine cells of gastrointestinal tract has shown multiple biological functions. When it flushes into liver through portal vein, VIP is bound to its receptor on the surface of hepatocytes. VIP receptor will innerize and degrade it. Finally, VIP will be cleared by lysosome. Therefore, liver is a crucial place to clear and inactivate overwhelming majority of VIP. We are interested in these espects: What effects do VIP and receptor in liver on liver hematopoiesis and migration? And what effects do VIP and receptor in liver on transdifferentiation of HSC
in liver? To answer these questions, we studied the direct effect of VIP on HSC biological function and behavior.
Objectives
In this research,we try to investigate:
1. The effects and mechanism of Vasoactive Intestinal peptide(VIP) on proliferation and colony forming unit(CFU) of Hematopoietic Stem Cells(HSC).
2. The effect of VIP on differentiation of HSC to blood cells.
3. The effect of VIP on differentiation of HSC to hepatic related cells to probe the possibility that VIP affect HSC transdifferentiation.
4. Quantitative alteration of VEP and its receptor in hepatic tissue in the course of rat liver development to study the migrative mechanism of fetal liver hematopoiesis.
5. Does VIP mediate the function of HSC via the corresponding receptors on HSC? What subtype of VIP receptors localized on HSC?
Methods
1. Human cord blood were collected, and to isolate mononuclear cells by Ficoll.
2. MACS assay was used to purify CD34+ cells from MNC, the purity of the CD34+ cells was evaluated by flow cytometry.
3. Colony Forming Unit(CFU) enumeration was used to assess the effect of VIP on proliferation of HSC.
4. Amplification culture was counted to judge the effect of VIP on CD34+ cells.
5. TNF-a, TGF-beta and AFP in cultured HSC and its supernatant were measured with ELJSA assay.
6. Different blood cells in different culture time of CD34+ cells were identified by Flow Cytometry(FCM).
7. Liver tissue markers on HSC, AFP, ALB and CK-19, were measured by
immunohistochemistry.
8. Western blot assay was used to detect the expression of ALB on HSC.
9. Nest RT-PCR was used to detect the expression of AFP mRNA and ALB mRNA on HSC, the product of ALB was chosen to measure the sequence.
10. SD rat liver tissues in different development phase were collected.
11. Biacore assay was used to detect the expression of receptor for VIP on human HSC and rat liver tissue.
12. Radioimmunologic assay was used to detect the concentration of VIP in rat liver development.
13. VIPR mRNA on human HSC and rat liver tissue was identified by RT-PCR.
Results
1. CFUs of CD34+ cells treated with VIP in the concentration range of 10~7 mol/L ~10-12 mol/L were significantly lower than that of control group (inhibition ratio > 26.97 ±13.72%), P<0.05. VIP at 10-8 mol/L showed the highest inhibition. With this concentration, the proliferative folds of CD34+ cells were 11.78±4.39. 16.71 ±2.98. 21.69±3.28 at the day 7, 10 and 14 separaterly. They were greatly lower than those of control group (20.13± 3.32. 25.64±3.81> 28.33±2.61), P<0.05.
2. VIP upregulated the concentration of TNF-a in HSC significantly, it was 149.15 pg/ml, 49% higher than control, P<0.05. VIP also markly increased the concentration of TGF-beta1 in HSC, it was 116.10 pg/ml,
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