Age-related changes in cerebral angiogenesis and fetal liver kinase-1 expression after cerebral ischemia/reperfusion

Cerebral angiogenesis in the early stages after cerebral ischemia injury is essential for the recovery of nerve function,in which fetal liver kinase-1（Flk-1）,as a major regulator of vasculogenesis and angiogenesis,plays a very important role.Microvessel density（MVD）was greater in an aged model group compared with the young sham operated group（P 〈 0.01）.MVD and the sum of the lumen area were decreased in the aged group at 1,3,6 and 12 days following ischemia/reperfusion（I/R）injury compared with the young model group（P 〈 0.05 and P 〈 0.01,respectively）.Flk-1 protein and mRNA expression was greater in the aged model group when compared with the young sham operated group（P 〈 0.01）.Flk-1 protein and mRNA expression was lower in the aged group at 1,3,6 and 12 days after I/R compared with the young model group（P 〈 0.01）.Flk-1 expression in aged rats attenuated rapidly,but was still maintained at relatively higher levels at 12 days following I/R in younger rats.The results suggest that angiogenesis was weakened after cerebral I/R in aged rats,and the mechanism of which might be correlated with attenuated expression of Flk-1 protein and mRNA.

EFFECT OF HEPATIC STIMULATOR SUBSTANCE (HSS) EXTRACTED FROM FETAL LIVER ON THE PROLIFERATION OF HUMAN ALEXENDER HEPATOMA CELLS

It's reported that hepatic stimulator substance (HSS) was extracte from the fetal liver of 4 - 6 months of fetus, and that the effect of HSS on the proliferation of human Alexender hepatoma cells was studied in this paper. The results showed that proliferation of Alexender cells varied with the amount of HSS in the culture medium, and the former was positively correlated with the latter significantly (P<0. 01). The study indicated that HSS from the fetal liver can stimulate the proliferation of human Alexender hepatoma cells.

Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells

瞄准：为了与胎儿的肝细胞(FLC ) 和可能性在合作文化调查间充质的干细胞(MSC ) 的 hepatocytic 区别膨胀，区分了 hepatocytic 房间。方法：MSC 被制动火箭与绿荧光灯的蛋白质(GFP ) 标记病毒的基因转导变异。同种细胞的显著 MSC 在用与干细胞补充的fibronectin涂的培养皿和媒介刺激条件的肝下面是也有教养的因素( SCF )， hepatocyte 生长因素( HGF )，表皮的生长因素( EGF )，和成纤维细胞生长因素 4 ( FGF-4 )独自一个，或在刚孤立的 FLC 的存在。在合作文化的房间被收获，并且 GFP+ 或 GFP- 房间用荧光被分开激活的房间排序。为肝 specific 标记 cytokeratin-18 (CK-18 ) 的反向的抄写聚合酶链反应(RT-PCR )( 法新社) ，白朊，和 alpha-fetoprotein 在不同房间人口被执行。结果：在指定文化条件下面，与 FLC co 有教养的老鼠 MSC 超过二个星期表示了白朊， CK-18，和 AFP-RNA。在 wk 3， MSC 失去了 hepatocytic 基因表示，可能由于 cocultured FLC 的增生。FLC 也在合作文化和一个很高的生长潜力显示出稳定的肝 specific 基因表情。结论：从骨髓的老鼠 MSC 能面对 FLC 在试管内区分 hepatocytic 房间，在合作文化的 MSC 的存在也为 FLC 的扩大和区别提供有益的环境。

Characterization of hepatic progenitors from human fetal liver during second trimester

AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. METHODS: EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin β1), CD49f (integrin α6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ (A, B, C) and class Ⅱ (DR) expression was studied by flow cytometry only. RESULTS: FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class Ⅰ, albumin and AFP but negative for HLA class Ⅱ (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. CONCLUSION: Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells

AIM: To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes. METHODS: Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC- assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone. RESULTS: The mRNA expression of α-fetoprotein, albumin (ALB), α-1-antitrypsin, and hepatocyte nuclear factor 4α was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storageand urea synthesis were also demonstrated. CONCLUSION: MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker

AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker. METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation. RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture. CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

Transplantation of fetal liver epithelial progenitor cells ameliorates experimental liver fibrosis in mice

AIM: To investigate the effect of transplanted fetal liver epithelial progenitor (FLEP) cells on liver fi brosis in mice. METHODS: FLEP cells were isolated from embryonal day (ED) 14 BALB/c mice and transplanted into female syngenic BALB/c mice (n = 60). After partial hepatectomy (PH), diethylnitrosamine (DEN) was administered to induce liver fibrosis. Controls received FLEP cells and non-supplemented drinking water, the model group received DEN-spiked water, and the experimental group received FLEP cells and DEN. Mice were killed after 1, 2, and 3 mo, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), and laminin (LN) in serum, and hydroxyproline (Hyp) content in liver were assessed. Alpha-smooth muscle actin (α-SMA) of liver was tested by immunohistochemistry. Transplanted male mice FLEP cells were identifi ed by immunocytochemistry for sry (sex determination region for Y chromosome) protein. RESULTS: Serum ALT, AST, HA, and LN were markedly reduced by transplanted FLEP cells. Liver Hyp content and α-SMA staining in mice receiving FLEP cells were lower than that of the model group, which was consistent with altered liver pathology. Transplanted cells proliferated and differentiated into hepatocytes and bile duct epithelial cells with 30%-50% repopulation in the liver fi brosis induced by DEN after 3 mo. CONCLUSION: Transplanted FLEP cells proliferate and differentiate into hepatocytes and bile duct epithelialcells with high repopulation capacity in the fiberized liver induced by DEN, which restores liver function and reduces liver fi brosis.

A New Hematopoietic Stimulating Activity Produced by Fetal Liver Cells

Human fetal liver cells were cultured in vitro for 12h and the supernatant(Fetal liver cell conditioned medium,FLCM)was collected.The effects of FLCM ongranulopoiesis were studied.The results show that when combined with colonystimulating factor(CSF),FLCM could significantly stimulate the proliferation of normalmyctoid progenitor cells(CFU-e),and increase ~3H-TdR incorporation into bone mar-row cells.The data suggest that FLCM contains a CSF synergistic activity.

Comprehensive Characterization and Global Transcriptome Analysis of Human Fetal Liver Terminal Erythropoiesis

The fetal liver(FL)is the key erythropoietic organ during fetal development,but knowledge on human FL erythropoiesis is very limited.In this study,we sorted primary erythroblasts from FL cells and performed RNA sequencing(RNA-seq)analyses.We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis.Notably,the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts(OrthoEs),suggesting the involvement of these pathways in enucleation.We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+cells.Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences,indicating impacts of the culture system on gene expression.Notably,the expression of lipid metabolism-related genes was increased in cultured erythroblasts.We further immortalized erythroid cell lines from FL and cord blood(CB)CD34+cells(FL-iEry and CB-iEry,respectively).FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs,but their enucleation ability was very low.Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity,indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate.Additionally,the expression of HBE1,HBZ,and HBG2 was up-regulated in FL-iEry compared with CB-iEry,and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1.Our study provides new insights into human FL erythropoiesis and rich resources for future studies.

Human CD34loCD133lo fetal liver cells support the expansion of human CD34hiCD133hi hematopoietic stem cells

We have recently discovered a unique CD34loCD133lo cell population in the human fetal liver （FL） that gives rise to cells in the hepatic lineage. In this study, we further characterized the biological functions of FL CD341~CD133~~ cells. Our findings show that these CD341~CD133I~ cells express markers of both endodermal and mesodermal lineages and have the capability to differentiate into hepatocyte and mesenchymal lineage cells by ex vivo differentiation assays. Furthermore, we show that CD34~~CD 133I~ cel Is express growth factors that are important for human hematopoietic stem cell （HSC） expansion： stem cell factor （SCF）, insulin-like growth factor 2 （IGF2）, C-X-C motif chemokine 12 （CXCL12）, and factors in the angiopoietin-like protein family. Co-culture of autologous FL HSCs and allogenic HSCs derived from cord blood with CD34loCD133lo cells supports and expands both types of HSCs.These findings are not only essential for extending our understanding of the HSC niche during the development of embryonic and fetal hematopoiesis but will also potentially benefit adult stem cell transplantations in clinics because expanded HSCs demonstrate the same capacity as primary cells to reconstitute the human immune system and mediate long-term hematopoiesis in vivo. Together,CD34loCD133lo cells not only serve as stem/progenitor cells for liver development but are also an essential component of the HSC niche in the human FL.

THE MECHANISM OF CLINICAL EFFECTIVENESS OF HUMAN FETAL LIVER CELL TRANSFUSION

During the development of human fetus, all organs consisting of various types of cells are in rapid growth. The significance of these cells and the chemical components involved, especially those of special functions in medical treatment of certain refractory diseases, have been noticed gradually. The questions as a step further are therefore raised, e.g. what is the mechanism underlying the clinical effectiveness and how to produce the active substances in large amount by biological engineering technique or chemical synthetic method, instead of using the crude preparation directly derived from fetus.

Isolation, characterization and culture of Thy1-positive cells from fetal rat livers

瞄准：为了调查 Thy1 是否在胎儿的肝认出卵形的房间并且描绘，有教养的 Thy1- 从 E14 老鼠肝选择了房间。方法：Thy1 人口被荧光分析激活的房间 sorter 分析。Thy1 积极房间用磁性的祷告被孤立。肝的标记被西方的弄污，免疫细胞化学和 RT-PCR 检测。结果：Thy1 积极的房间的百分比在胎儿的老鼠肝(E13-E16 ) 的早开发期间减少了。E14 胎儿的肝包含了 7.8% Thy1 积极房间， 61% 为 alpha-fetoprotein (法新社) 和 25% 表示白朊是积极的。Thy1+ 人口表示了卵形的房间标记 c 工具包和 CXCR4，肝充实抄写的因素 HNF1alpha 和 HNF6， hepatocytic 标记白朊，法新社和 cytokeratin 18，并且胆汁的标记 cytokeratin 19。Thy1- 选择了房间形成的仅仅间充质的殖民地什么时候骨胶原上并且在包含浆液的媒介的 plated。选择房间能形成为 HNF1alpha 积极的肝的殖民地的 Thy1， HNF6，白朊，法新社， cytokeratin 18， cytokeratin 19 并且肝糖，当在浆液在 STO 喂食器层上成长时免费媒介。结论：为 Thy1 积极的卵形的房间在早肝是在场的胚胎的阶段。

Fetal liver： an ideal niche for hematopoietic stem cell expansion

Fetal liver(FL) is an intricate and highly vascularized hematopoietic organ, which can support the extensive expansion of hematopoietic stem cells(HSCs) without loss of stemness, as well as of the downstream lineages of HSCs. This powerful function of FL largely benefits from the niche(or microenvironment), which provides a residence for HSC expansion. Numerous studies have demonstrated that the FL niche consists of heterogeneous cell populations that associate with HSCs spatially and regulate HSCs functionally. At the molecular level, a complex of cell extrinsic and intrinsic signaling network within the FL niche cells maintains HSC expansion. Here, we summarize recent studies on the analysis of the FL HSCs and their niche, and specifically on the molecular regulatory network for HSC expansion. Based on these studies, we hypothesize a strategy to obtain a large number of functional HSCs via 3 D reconstruction of FL organoid ex vivo for clinical treatment in the future.

Hepatic reconstruction from fetal porcine liver cells using a radial flow bioreactor

AIM: To examine the efficacy of the radial flow bioreactor (RFB) as an extracorporeal bioartificial liver (BAL) and the reconstruction of liver organoids using embryonic pig liver cells. METHODS: We reconstructed the liver organoids using embryonic porcine liver cells in the RFB. We also determined the gestational time window for the optimum growth of embryonic porcine liver cells. Five weeks of gestation was designated as embryonic day (E) 35 and 8 wk of gestation was designated as E56. These cells were cultured for one week before morphological and functional examinations. Moreover, the efficacy of pulsed administration of a high concentration hepatocyte growth factor (HGF) was examined. RESULTS: Both cell growth and function were excellent after harvesting on E35. The pulsed administration of a high concentration of HGF promoted the differentiation and maturation of these fetal hepatic cells. Microscopic examination of organoids in the RFB revealed palisading and showed that bile duct-like structures were well developed, indicating that the organoids were mini livers. Transmission electron microscopy revealed microvilli on the luminal surfaces of bile duct-like structures and junctional complexes, which form the basis of the cytoskeleton of epithelial tissues. Furthermore, strong expression of connexin (Cx) 32, which is the main protein of hepatocyte gap junctions, was observed. With respect to liver function, ammonia detoxification and urea synthesis were shown to be performed effectively. CONCLUSION: Our system can potentially be applied in the fields of BAL and transplantation medicine.

Partial isolation and identification of hepatic stimulator substance mRNA extracted from human fetal liver

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Effects of restrictions on maternal feed intake on the immune indexes of umbilical cord blood and liver Toll-like receptor signaling pathways in fetal goats during pregnancy

Background: Liver has important immune function during fetal development and after birth.However,the effect of maternal malnutrition on immune function of the fetal liver is rarely reported.In this study,twelve pregnant goats(Xiangdong black goat,at d 45 of gestation) were assigned to the control group(fed 100% of nutritional requirements) and the restriction group(fed 60% of the intake of the control group) during gestation from d 55 to100.Fetal goats were harvested at d 100 of gestation and immune indexes and amino acid profiles of the umbilical cord blood and liver Toll-like receptors(TLRs) signaling pathways were measured.Results: Maternal body weight in the restriction group was lower than the control group(P < 0.05).Maternal feed intake restriction decreased(P < 0.05) heart weight,heart index,alkaline phosphatase and serum amyloid protein A in the umbilical cord blood(UCB).Moreover,only histidine was decreased in the restricted group(P = 0.084),and there were no differences in other amino acids contents in the UCB between the two groups(P > 0.05).The TLR2 and TLR4 mRNA expression in the fetal liver in the restriction group was greater(P < 0.05) than that in the control group.Furthermore,the mRNA expression levels of myeloid differentiation primary response 88(MyD88),TNF receptor associated factor 6,nuclear factor kappa B subunit 1,NFKB inhibitor alpha,IFN-β,TGF-β,TNF-α and IL-1β in the restricted group were upregulated(P < 0.05),and the expression of TLR3(P = 0.099) tended to be higher in the restricted group.However,protein levels of TLR2,TLR4,IκBα,phosphorylated IκBα,phosphorylated IκBα/total IκBα,TRIF and MyD88 were not affected(P > 0.05) by maternal intake restriction.Conclusions: These results revealed that the restriction of maternal feed intake influenced the development of heart and hepatic protein synthesis at the acute phase of fetal goats and upregulated the mRNA expression of genes involved in MyD88-dependent signaling pathways and of target cytokines.

Renewal and preliminary study of expressed sequence tags database on human fetal liver aged 22 wk of gestation

With the developments of international human transcriptome data and our ESTs of human fetal liver aged 22 weeks (wk) of gestation (HFL22w), the former research must be renewed. In this work, the EST data were firstly clustered by blasting against the ESTs of HFL22w, UniGene, DoTS, MGC and Twinscan-predicted human transcriptome. Then, after EST assembly and gene identification, the known genes were classified by GO (gene ontology), and the unknown genes were predicted by Pfam and ScanProsite to clarify their functions. In the end, the relations of 5 tissues including fetal liver, adult liver, bone marrow, thymus and lymph node that possess hemopoiesis or can indicate fetal liver characteristics were analyzed by hierarchical clustering. The results show that: (i) By comparing the 5 newest human transcriptome databases, we can largely reduce the probability that the ESTs belonging to unconnected parts of one gene were probably divided into different clusters, so it is recommended to blast against the newest databases when clustering EST data; (ii) some previous unknown ESTs had been identified as function-known genes, and 1379 genes were identified as fully new sequences possessed in our lab; (iii) through GO classification, we got a rough understanding of HFL22w, and obtained 6 cell migration genes and 6 hemopoiesis genes; (iv) prediction of gene function had enabled us to obtain 277 profiles, among them, there are 5 categories distributed in more than 10 genes; (v) five tissue relations analyzed by hierarchical clustering are related to their functions; (vi) We have built the world's largest EST database on HFL22w. Renewal and preliminary analysis of EST database on HFL22w will help to understand hemopoiesis and cell migration mechanism, and promote future research on human fetal liver.

Investigation on Hepatopoietin and Other Novel Genes from Human Fetal Liver

The aim of this study is to discover the molecular mechanism of the 22-week gestated human fetal liver (HFL) which rarely displays both hematopoietic and hepatic functions.Based on large-scale cDNA library sequencing and bioinformatic analysis,the largest gene expression profile of human fetal liver in the world was successfully established.A set of gene clusters func- tionally related to the liver development,hepatocarci- nogenesis and hematopoiesis have been identified.This is for the first time that we could panoramically under- stand the molecular mechanism of the dual functions of human fetal liver.Moreover,201 unrecorded human homologous genes and 609 novel genes have been iden- tified and annotated,which accounting for more than 7% of the known human genes in 2001.In the recent human genome annotation map (human genome build 35. 1 ), 45 genes were nominated based on this study. In addition, we have characterized a set of gene fami- lies represented by hepatopoietin (HPO), Semaphorin, LSECtin and ARFGAP.Two distinctive novel pathways, 'extracellular HPO→HPOreceptor→EGF receptor→Raf→MEK→MAPK' for autocrine and 'intracellular HPO→JAB1→c-JUN(AP-1)' for intracrine of HPO, an unusual cytokine functioned in the regeneration of liver, has been reported for the first time, which have shed new lights on the study of the signal transduction of the entire HPO family.We have also demonstrated that HPO could act as a FAD thioloxidase and that only its intracrine pathway is dependent on the enzymatic activi- ty. It is also known for the first time that the enzyme activity is critically important for the cytokine HPO.Re- garding the regulation of the gene expression of HPO,it was demonstrated that HPO promoter includes a nega- tive regulatory element and a core promoter (comprises an initiator and its flanking three tandem IFE elements). Furthermore,two novel members of Semaphorin family, SEMA6C and SEMA6D,were cloned and shown to be able to determine the orientation of the cell growth.We have also discovered and characterized a novel lectin family including LSECtin, CD23,DC-SIGN and DC- SIGNR.The function of LSECtin was also defined to be important in adhesion of the cells.In addition,the first human member of ARFGAP family was cloned and shown to regulate protein secretion.The publications based on this study have been cited for 145 times by SCl journals before 2005.This study has provided im- portant original data for the annotation of human genome and establishment of human transcriptome.It also played an important role in Chinese national achievement of cloning and annotation of the 10% human cDNAs pro- ject and set up the corner-stone for the leading role of China in the international 'Human Liver Proteome Pro- ject'.

Universal RNA editing in a human liver at the fetal stage

It is known that RNA editing occurs in human cells, which can change the information transmission from DNA to RNA and proteins. Most previous studies have focused on editing of the mRNAs. Here we reported that several kinds of RNAs, including miRNA, rRNA, mRNA, miscRNA and unknown RNA, exhibited base editing in a human fetal liver. Several editing types are displayed. Our data reveals that RNA editing may occur in different species of RNAs.

Effect of Bushen Yiqi Huoxue Recipe on Placental Vasculature in Pregnant Rats with Fetal Growth Restriction Induced by Passive Smoking

Interactions of vascular endothelial growth factor （VEGF） with receptors VEGFR1/Fltl and VEGFR2/Flk1, and those of angiopoietins （Ang-1, Ang-2） with receptor Tie2 play important roles in placental angiogenesis. This study investigated vascular morphology and expression of these angiogenic factors in rat placenta on the day 15, 18, 21 of gestation （D 15, D 18 and D21）. The rats were randomly assigned into 3 groups： normal group, model group [fetal growth restriction （FGR） model], and Bushen Tqi Huoxue （BYHR） recipe treatment group （BYHR group, the pregnant rats with FGR were treated with BYHR recipe）. Morphological analysis indicated that during initial villous formation, fetal nucle- ated erythrocytes （FNEs） appeared in maternal blood sinus （MBS）. Subsequently, FNEs were sur- rounded by endothelial cells to form fetal capillary （FC） and then by trophoblast cells to form villi. As pregnancy proceeded, FC density increased progressively with increasing endothelial identification staining （EIS） in normal and BYHR groups. Whereas, villous formation was suppressed, normal in- crease in FC density was impaired and EIS was weakened in model group. Quantitative PCR analysis showed that VEGF and Flkl mRNA increased over gestation in all groups, indicating that VEGF might play a pivotal role in FC growth during late gestation. VEGF mRNA was increased on D15, while de- creased on D21 in model group as compared with normal group and BYHR group. Immunohistochemi- cally, Ang-2 protein was highly expressed in FNEs, gradually disappeared as villi matured, and decreased over gestation in all groups, indicating that Ang-2 might play a pivotal role in villous formation, which was further supported by decreased Ang-2 mRNA and protein expression in model group on D 15. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio increased from D15 to D18 in all groups as placenta matured. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio were decreased on D18 in model group as compared with normal and BYHR groups, indicating delayed maturity of FGR placenta. Alterations in angiogenic factors may result in altered placental vasculature and cause placental insufficiency. BYHR recipe could balance the angiogenic factors to promote the formation and maturation of FGR placental vasculature.