Establishment of the First National Standard for Neutralizing Antibodies against SARS-CoV-2 XBB Variants.

Neutralizing antibodies (NtAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are indicators of vaccine efficacy that enable immunity surveillance. However, the rapid mutation of SARS-CoV-2 variants prevents the timely establishment of standards required for effective XBB vaccine evaluation. Therefore, we prepared four candidate standards (No. 11, No. 44, No. 22, and No. 33) using plasma, purified immunoglobulin, and a broad-spectrum neutralizing monoclonal antibody. Collaborative calibration was conducted across nine Chinese laboratories using neutralization methods against 11 strains containing the XBB and BA.2.86 sublineages. This study demonstrated the reduced neutralization potency of the first International Standard antibodies to SARS-CoV-2 variants of concern against XBB variants. No. 44 displayed broad-spectrum neutralizing activity against XBB sublineages, effectively reduced interlaboratory variability for nearly all XBB variants, and effectively minimized the geometric mean titer (GMT) difference between the live and pseudotyped virus. No. 22 showed a broader spectrum and higher neutralizing activity against all strains but failed to reduce interlaboratory variability. Thus, No. 44 was approved as a National Standard for NtAbs against XBB variants, providing a unified NtAb measurement standard for XBB variants for the first time. Moreover, No. 22 was approved as a national reference reagent for NtAbs against SARS-CoV-2, offering a broad-spectrum activity reference for current and potentially emerging variants.

Establishment of national standard for anti-SARS-Cov-2 neutralizing antibody in China: The first National Standard calibration traceability to the WHO International Standard.

Neutralizing antibody (NtAb) levels are key indicators in the development and evaluation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. Establishing a unified and reliable WHO International Standard (IS) for NtAb is crucial for the calibration and harmonization of NtAb detection assays. National and other WHO secondary standards are key links in the transfer of IS to working standards but are often overlooked. The Chinese National Standard (NS) and WHO IS were developed by China and WHO in September and December 2020, respectively, the application of which prompted and coordinated sero-detection of vaccine and therapy globally. Currently, a second-generation Chinese NS is urgently required owing to the depletion of stocks and need for calibration to the WHO IS. The Chinese National Institutes for Food and Drug Control (NIFDC) developed two candidate NSs (samples 33 and 66-99) traced to the IS according to the WHO manual for the establishment of national secondary standards through a collaborative study of nine experienced labs. Either NS candidate can reduce the systematic error among different laboratories and the difference between the live virus neutralization (Neut) and pseudovirus neutralization (PsN) methods, ensuring the accuracy and comparability of NtAb test results among multiple labs and methods, especially for samples 66-99. At present, samples 66-99 have been approved as the second-generation NS, which is the first NS calibrated tracing to the IS with 580 (460-740) International Units (IU)/mL and 580 (520-640) IU/mL by Neut and PsN, respectively. The use of standards improves the reliability and comparability of NtAb detection, ensuring the continuity of the use of the IS unitage, which effectively promotes the development and application of SARS-CoV-2 vaccines in China.

Potent Anti-SARS-CoV-2 Efficacy of COVID-19 Hyperimmune Globulin from Vaccine-Immunized Plasma.

Coronavirus disease 2019 (COVID-19) remains a global public health threat. Hence, more effective and specific antivirals are urgently needed. Here, COVID-19 hyperimmune globulin (COVID-HIG), a passive immunotherapy, is prepared from the plasma of healthy donors vaccinated with BBIBP-CorV (Sinopharm COVID-19 vaccine). COVID-HIG shows high-affinity binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, the receptor-binding domain (RBD), the N-terminal domain of the S protein, and the nucleocapsid protein; and blocks RBD binding to human angiotensin-converting enzyme 2 (hACE2). Pseudotyped and authentic virus-based assays show that COVID-HIG displays broad-spectrum neutralization effects on a wide variety of SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), and Omicron (B.1.1.529) in vitro. However, a significant reduction in the neutralization titer is detected against Beta, Delta, and Omicron variants. Additionally, assessments of the prophylactic and treatment efficacy of COVID-HIG in an Adv5-hACE2-transduced IFNAR-/- mouse model of SARS-CoV-2 infection show significantly reduced weight loss, lung viral loads, and lung pathological injury. Moreover, COVID-HIG exhibits neutralization potency similar to that of anti-SARS-CoV-2 hyperimmune globulin from pooled convalescent plasma. Overall, the results demonstrate the potential of COVID-HIG against SARS-CoV-2 infection and provide reference for subsequent clinical trials.

The first Chinese national standards for SARS-CoV-2 neutralizing antibody.

In order to meet the domestic urgent needs of evaluating the immunogenicity of vaccines and the potency testing of therapeutic antibody products against coronavirus disease 2019 (COVID-19), the first Chinese national standards for SARS-CoV-2 neutralizing antibody were established. The potency and stability of the candidate standards were determined by neutralization assay and accelerated degradation study. The stability studies showed that the standards were stable in the short-term. The collaborative study showed that the candidate standards could reduce the variations in neutralization titers between labs and thus improve comparability of neutralizing antibody measurements. Sample 22 has been approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese National Standard for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralizing antibody, with an assigned potency of 1,000 units per milliliter (U/ml). This standard will contribute to the standardized assessment of the quality and efficacy of vaccines and therapeutics for COVID-19 in China.

In vivo study of novelly formulated porcine-derived fibrinogen as an efficient sealant.

Fibrinogen has been used as surgical sealant in the clinical setting for decades. The application of human plasma-derived fibrinogen is limited due to high cost and the risk of prion and virus infection. We developed a novel arginine-formulated fibrinogen from cryoprecipitates of porcine plasma. This porcine-derived fibrinogen exhibited excellent stability during sterilization and better hemostatic efficacy than a leading commercial hemostatic product in a nonlethal hemorrhage model. Therefore, it has the potential to be more economical and readily available while having a decreased risk of human blood-borne pathogen transmission.

Differentiation of adipose-derived adult stem cells into epithelial-like stem cells.

Adipose-derived adult stem (ADAS) cells can be easily obtained in large quantities. Previous studies have suggested that all-trans-retinoic acid (ATRA) plays an important role in the differentiation of mesenchymal stem cells toward an epithelial lineage. In order to verify that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells, ADAS-cells were isolated and cultured. They were induced to differentiate toward an epithelial lineage in vitro. Differentiated epithelial cells were assayed as to whether they retain characteristics of stem cells by RT-PCR and cell cycle stage analysis, and were further induced to differentiate toward an osteogenic lineage. RT-PCR analysis revealed that no CK5, CK10 or CK19 mRNA was detected in ADAS-cells, CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10. After induction, the expression of CK19 was observed by immunofluorescent staining. Positive staining with alkaline phosphatase (ALP) and Von Kossa staining verified that differentiated epithelial cells still had potential to further differentiate toward an osteogenic lineage. These experiments provide proof that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells.

The 9L(LUC)/Wistar rat glioma model is not suitable for immunotherapy.

The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9L(LUC) glioma cells syngenic to Fischer 344 (F344) rats. Bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9L(LUC)/F344 rats, and tumor regression was found in some 9L(LUC)/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9L(LUC)/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9L(LUC)/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo.

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.

Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer.

Abnormal activation of the Sonic hedgehog (SHH) pathway has been described in a wide variety of human cancers and in cancer stem cells (CSCs), however, the role of SHH pathway in gastric CSCs has not been reported. In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells (CSLCs) from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2 x 104 tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.

Hepatogenesis of adipose-derived stem cells on poly-lactide-co-glycolide scaffolds: in vitro and in vivo studies.

Human adipose-derived stem cells (hASCs) have been shown to be multipotent and could be induced into various cell types, which make them the ideal cell source for cell therapy or tissue engineering. However, differentiation of ASCs into hepatocytes on three-dimensional scaffold, an important part of tissue engineering, has not been reported. In this study, to investigate the hepatogenesis of ASCs on porous poly-lactide-co-glycolide (PLGA) scaffolds, we loaded hASCs on these scaffolds. The cell-scaffold complex was implanted into the peritoneal cavity of 70% hepatectomized rats with or without 14 days of induction in hepatic inducing medium. Our results indicated that hASCs cultured on the PLGA scaffolds in the hepatic inducing medium proliferated more efficiently and could be induced into cells with hepatocyte-like phenotypic and functional properties. In vivo studies showed that induced hASCs on PLGA scaffolds survived and maintained hepatic phenotype and function for at least 14 days after implantation; moreover, noninduced hASCs on PLGA scaffolds expressed human albumin 14 days after transplantation. Collectively, these results suggest that porous PLGA scaffolds are suitable for the hepatogenesis of hASCs. These findings might be helpful in the application of hASC-based tissue engineering for liver disease therapy.

[Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro].

OBJECTIVE: To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. METHODS: hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. RESULTS: The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. CONCLUSION: hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

[Effects of different culture conditions on isolation and expansion of stem cells from second-trimester amniotic fluids].

OBJECTIVE: To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids. METHODS: Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16 - 24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008. Amniotic fluids (10 - 20 ml) samples were collected and each was cultured under different conditions or groups. (1) Low-glucose DMEM (LD) medium supplemented with 10% of fetal bovine serum (group of 10%FBS); (2) LD medium with 20% of FBS (group of 20%FBS); (3) LD medium with 15% of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF); (4) LD medium with 10% of FBS as well as the culture plate coated with gelatin (group of gelatin). The effects of different conditions were evaluated by comparing the number of primary colonies, the cell morphology and the ability of expansion. The isolated stem cells were identified by flow cytometry, RT-PCR and differentiation ability to adipocyte. RESULTS: (1) The success rates of primary culture of the group of 10%FBS, 20%FBS, bFGF and gelatin were 60%, 73%, 73% and 60% respectively (P > 0.05). The numbers of colonies were 0.9 +/- 0.5, 2.6 +/- 1.5, 2.9 +/- 1.5, 1.1 +/- 0.8 (P < 0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF); among the primary colonies, fibroblast-like colonies accounted for 46%, 49%, 64%, 44% respectively (P > 0.05). (2) The second passage cells obtained from all of these four groups could differentiate into adipocyte after induction. (3) In the group of bFGF, stem cells were isolated from 5 samples and expanded to nearly 10(7) cells after 5 passages (P < 0.01 compared with other groups). (4) Karyotype were normal in all samples. (5) Stem cells from bFGF group showed positive expression of SSEA-4, Oct-4 and Nanog gene detected by flow cytometry and RT-PCR. CONCLUSION: Stem cells can be isolated from second-trimester amniotic fluids; moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.

[Repair of swine full-thickness cutaneous deficiency by autogenic BMSCs compounded with collagen membrane].

OBJECTIVE: To supply references to tissue-engineered skin clinical applications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous deficiency. METHODS: Twenty mL bone marrow were obtained respectively from 4 swine, autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendon treated by means of chemically cross-linked was made 5 cm diameter collagen I (Col I) membrane. The 2 x 10(7)/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation, then the tissue-engineered skin was constructed. The five full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midline two sides of swine. The tissue-engineered skin were implanted in the experimental group, while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation, the two swine wound surface healing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation, the experimental group were observed with fluorescence microscopy and staining for glycogen. RESULTS: After 3 weeks of implantation, the wound surface of control group were observed nigrescene, scab and putrescence, and after 8 weeks of implantation, also evident putrescence and scar. The wound surface of experiment group was alive after 3 weeks implantation, appearance was leveled off and flexible without evident scar. The wound surface recovered well after 8 weeks of implantation, wound surface healing rate was significantly difference between the two groups (P < 0.01). After 3 weeks of implantation, control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. The experimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation, the experimental group and control group were emerged normal skin structure. After 3 weeks of implantation in control group, a lot of neutrophilic granulocytes and fibroblasts were noticed, but no epidermal structure was observed under TEM. In the experimental group, a lot of epidermal cells were observed, dermatome connection among epidermal cells and hemidesmosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group, blood capillary endothelial cells were noticed. Furthermore, considerable collagen fiber deposit was found in the surrounding tissue of fibroblasts. After 3 weeks of implantation, BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by fluorescence microscopy. CONCLUSION: Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in application of repairing swine full-thickness cutaneous deficiency.

[Analysis of properties of collagen membranes before and after crosslinked].

OBJECTIVE: To compare the properties of collagen membranes before and after crosslinked and to establish the foundation of application of collagen membranes. METHODS: Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophilization. And then the biocharacteristics of the membranes before and after glutaraldehyde crosslinked were compared. BMSCs separated from volunteer's bone marrow were seeded on collagen membranes before and after crosslinked by 2 x 10(3) in 100 microL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). RESULTS: The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecular weight of main bands was more than 66.2 x 10(3), the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% praline and 10.18% hydroxyproline. No tryptophan was found. Before crosslinked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3,400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crosslinked collagen membranes or on after-crosslinked ones. CONCLUSION: As biomaterial scaffolds, after crosslinked collagen membranes were better than before-crosslinked ones.

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells.

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% +/- 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering.

[Protective effect of sodium pyruvate on ischemia/reperfusion injury of rats subjected to hemorrhagic shock].

AIM: To study the protective effect of sodium pyruvate on ischemia/reperfusion injury following hemorrhagic shock. METHODS: Rat models of hemorrhagic shock were built up. When the shed blood was infused, the rats were also randomly provided by one of normal saline, glutathione and sodium pyruvate. Rats were killed 3 hours after the reperfusion, the activity of plasma lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT), the level of tissue malondialdehyde (MDA) and the activity of tissue myeloperoxidase (MPO) were detected. Biopsy specimens were obtained to investigate morphological changes of the myocardial, hepatic, lung and renal tissue. RESULTS: The activity of plasma LDH and GOT, the level of MDA of hepatic, lung and renal tissue and the activity of MPO of myocardial, lung and renal tissue decreased remarkably in group given sodium pyruvate compared with group given normal saline, and the effect of group given sodium pyruvate was more remarkable than group given glutathione. CONCLUSION: These data support the view that sodium pyruvate shows protective effect on ischemia/reperfusion injury following hemorrhagic shock. It is possibly relevant to scavenging of oxygen free radicals, reduction of neutrophil, and anti-inflammatory response.

[Differentiation of bone marrow derived Thy-1+ beta2M- cells into liver cells in AA induced liver injury micro-environment].

OBJECTIVE: To investigate the differentiation of bone marrow derived Thy-1+ beta2M- cells (BDTCs) into liver cells in allyl alcohol (AA) induced liver injury micro-environment. METHODS: BDTCs of male F344 rats were isolated by two-step magnetic separation system (MACS) technique, and infused intraportally into female recipients after labeling with PKH26. Thirty recipients were divided randomly into 3 groups: (1) AA-injured liver + BDTCs infusion, (2) normal liver + BDTCs infusion and (3) AA-injured liver + NS infusion (control). Blood biochemical examination, fluorescence labeled cellular localization, Y-chromosome sry gene in-situ hybridization and immunohistochemistry were carried out to evaluate BDTCs distribution, differentiation and proliferation in recipients's livers after different intervals. RESULTS: Fluoromicroscopy and in situ hybridization suggested that BDTCs of donors were interspersed in pieces and cords among the necro-periportals induced by AA; immunohistochemistry indicated that those implanted cells expressed OV-6, AFP, CK19 and albumin successively, while positive cells were hardly seen in the normal liver + BDTCs infusion group. Compared with the controls, the blood biochemical restitution was more rapid in group (1), (9.8 d +/- 3.1 d vs. 13.7 d +/- 4.2 d). CONCLUSION: The injury micro-environment induced by AA facilitates BDTCs integration with hepatic cell plates and differentiation into mature liver cells. BDTCs differentiation into liver cells might accelerate endogenous liver cell regeneration and reparation.

[The expression of fusion protein GST-GP302 in E.coli and its preparation of rabbit anti-serum].

AIM: To express fusion protein of GST and vWf binding domain(GP302) of platelet GPIbalpha in E.coli and its preparation of rabbit anti-serum. METHODS: GP302 gene was inserted into pGEX-4T-1. The recombinant vector was identificated by restriction endonuclease digestion analysis. Fusion protein GST-GP302 was expressed in E.coli via IPTG induction. The rabbit antibody against GST-GP302 was prepared by using renatured GST-GP302 as immuneogen and the specificity of polyclonal antibody was identified by Western blot. RESULTS: The restriction endonuclease digestion analysis of recombinant plasmid demonstrated that the GP302 gene had been exactly inserted into pGEX-4T-1. SDS-PAGE analysis showed that the ralative molecular mass(M(r)) of the fusion protein was about 59 000. ELISA analysis proved that the titer of rabbit serum against GST-GP302 was 10(-5). The polyclonal antibody specifically bound to purified platelet GPIbalpha. CONCLUSION: The preparation of polyclonal antibody against GP302 peptide provides an usefal reagent for the detection of platelet GPIbalpha.