Role of TRPV1 in susceptibility to PTZ-induced seizure following repeated hyperthermia challenges in neonatal mice.

This study was designed to investigate the role of experimental febrile seizures in the induction of generalized clonic seizures and the involvement of heat-sensitive channel TRPV1. Pentylenetetrazol-induced clonic seizure was used as the seizure model, and Trpv1 gene knock-out and wild-type C57/BL6 mice were used as experimental subjects. Electroencephalograph and seizure behavior were recorded for the evaluation of the severity of seizures. Increased frequency of the experimental febrile seizures facilitated PTZ-induced generalized clonic seizures. Trpv1 gene deficiency decreased the properties of generalized clonic seizure. The intensity of experimental febrile seizures reduced the threshold to generalized clonic seizure, and Trpv1 gene deficiency decreased the susceptibility to PTZ-induced seizures following early-life hyperthermia challenges in mice.

Migration of dorsal aorta mesenchymal stem cells induced by mouse embryonic circulation.

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid-gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45(-)CD31(-)Flk-1(-)CD44(+)CD29(+)), expressed perivascular markers (α-SMA(+)NG2(+)PDGFRß(+)PDGFRα(+)), and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5-E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA-MSCs through platelet-derived growth factor (PDGF) receptor-, transforming growth factor-beta receptor-, but not basic fibroblast growth factor receptor-mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood- or PDGF-mediated migration of DA-derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis.

Maternal inappropriate calcium intake aggravates dietary-induced obesity in male offspring by affecting the differentiation potential of mesenchymal stem cells.

BACKGROUND: The effects of inappropriate dietary calcium intake in early life on later obesity have not been fully elucidated. AIM: To raise the mechanism of maternal calcium intake on the multi-differentiation potential of mesenchymal stem cells among their male offspring. METHODS: Four-week-old female C57BL/6N mice were fed by deficient, low, normal and excessive calcium reproductive diets throughout pregnancy and lactation. Bone MSCs (BMSCs) were obtained from 7-day-old male offspring to measure the adipogenic differentiation potential by the Wnt/ß-catenin signaling pathway. The other weaning male pups were fed a high-fat diet for 16 wk, along with normal-fat diet as the control. Then the serum was collected for the measurement of biochemical indicators. Meanwhile, the adipose tissues were excised to analyze the adipocyte sizes and inflammatory infiltration. And the target gene expressions on the adipogenic differentiation and Wnt/ß-catenin signaling pathway in the adipose tissues and BMSCs were determined by real-time reverse transcription polymerase chain reaction. RESULTS: Compared with the control group, maternal deficient, low and excessive calcium intake during pregnancy and lactation aggravated dietary-induced obesity, with larger adipocytes, more serious inflammatory infiltration and higher serum metabolism indicators by interfering with higher expressions of adipogenic differentiation (PPARγ, C/EBPα, Fabp4, LPL, Adiponectin, Resistin and/or Leptin) among their male offspring (P < 0.05). And there were significantly different expression of similar specific genes in the BMSCs to successfully polarize adipogenic differentiation and suppress osteogenic differentiation in vivo and in vitro, respectively (P < 0.05). Meanwhile, it was accompanied by more significant disorders on the expressions of Wnt/ß-catenin signaling pathway both in BMSCs and adulthood adipose tissues among the offspring from maternal inappropriate dietary calcium intake groups. CONCLUSION: Early-life abnormal dietary calcium intake might program the adipogenic differentiation potential of BMSCs from male offspring, with significant expressions on the Wnt/ß-catenin signaling pathway to aggravate high-fat-diet-induced obesity in adulthood.

Is It Necessary to Perform the Pharmacological Interventions for Intrahepatic Cholestasis of Pregnancy? A Bayesian Network Meta-Analysis.

BACKGROUND AND OBJECTIVE: Although many meta-analyses have evaluated the pharmacotherapy of intrahepatic cholestasis of pregnancy (ICP) and recommended ursodeoxycholic acid (UDCA) as an effective treatment, the defect of the pair-wise analyses and the mixture of the control group made the outcome uncertain and unclear. We aimed to employ Bayesian network meta-analysis (NMA) to compare the maternal and fetal outcomes after UDCA, S-adenosylmethionine (SAMe) mono-therapy or the combination treatment of these two drugs for ICP patients. METHODS: Multiple electronic database searches were conducted for articles published up to 1 September 2018. The relevant information was extracted from the published reports with a predefined data extraction sheet, and the risk of bias was assessed with the Cochrane risk-of-bias tool. Poisson Bayesian network meta-analysis was employed to identify the synthesized evidence from the relevant trials, with reporting hazard risks (HRs) and 95% credible intervals (CrIs). RESULTS: The pooled outcomes of the 13 randomized controlled trials (RCTs) with 625 participants indicated that none of the three regimens can significantly improve maternal and fetal outcomes. CONCLUSION: This NMA of the RCTs clarified that the current intervention has no favorable effect on pruritus and other symptoms in ICP patients.

[Comparative study for detecting micrometastases of lymph nodes in patients with lung cancer].

OBJECTIVE: To set up a new method to detect occult micrometastases of lymph nodes in patients with no-small-cell lung cancer (NSCLC). METHODS: We had detected 195 lymph node samples taken from 25 patients with NSCLC during the operations. Each lymph node sample was divided into two same parts in size. The one half part of lymph node should be examined by conventional histopathologic examination and immunohistochemical (IHC) staining. All the remaining lymph node samples of each patient should be mixed together for the reverse transcriptase-polymerase chain reaction (RT-PCR) if they located in the same region. As long as the presence of metastatic tumor in one lymph node was found by H&E staining, all other lymph node samples in the same region should not be detected by IHC staining or RT-PCR techniques. RESULTS: Frozen tissue sections of 135 lymph nodes that were staged as free of metastases by conventional histopathologic examination were screened by IHC staining. 31 lymph nodes showed single cell or cells clusters. Of 39 groups mixed regional lymph nodes which were diagnosed to be devoid of metastases by conventional histopathologic examination, 11 groups were found to have positive reactions to cytokeratin 19-mRNA by RT-PCR. There was a correlation between IHC staining and RT-PCR for detection of nodal micrometastases of NSCLC (U = 7.682, P = 0.0001). CONCLUSION: IHC staining analysis can facilitate the detection of occult micrometastases in lymph nodes of NSCLC, and its assessment of nodal micrometastases can provide a refinement of TNM stage for partial patients with stage I to II. RT-PCR has the same value as IHC staining in detection of lymph nodal micrometastases. RT-PCR technique can reduce expense of the detecting micrometastases in lymph nodes.

[MicroRNA-3963 Promotes Adipogenic Differentiation of Mouse-Derived Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of MicroRNA-3963(miR-3963) on the adipogenic differentiation of mouse bone-derived mesenchymal stem cells(MSC). METHODS: MSCs were isolated from C57BL/6 mice bone fragment and transfected with miR-3963 mimic, miR-3963 inhibitor and negative control. The expression of miR-3963 and transfection efficiency were detected by q-PCR. These transfected cells were induced to adipocytes and stained with oil red O after 14 days culture. q-PCR and Western blot were used to detect the expression of adipogenic differentiation marker genes C/EBPα and PPARγ at transcriptional level and protein level. RESULTS: The results of q-PCR revealed that miR-3963 expression level was up-regulated after transfection with miR-3963 mimic (P<0.0001), and down-regulated after transfection with miR-3963 inhibitor (P<0.0001). After oil red staining, overexpression of miR-3963 in MSCs could promote the formation of lipid droplet. The q-PCR and Western blot analyses showed the significant increase of expression of adipogenic marker genes C/EBPα and PPARγ in MSC transfected with miR-3963 mimic. Additionally, compared with the control group, miR-3963 inhibitor could decrease adipogenic differentiation of MSC. CONCLUSION: miR-3963 can regulate and promote adipogenic differentiation of mouse bone-derived MSC.

[Clinicopathological study on nodal molecular staging of non-small-cell lung cancer].

OBJECTIVE: To investigate the practicability of detecting the micrometastasis in lymph nodes of no-small-cell lung cancer (NSCLC) by means of reverse transcriptase-polymerase chain reaction (RT-PCR). METHODS: Regional lymph node samples were collected during operation from 25 patients with NSCLC randomly selected. The lymph node sample from each patient was divided into 2 groups: lymph nodes of hilum of lung and of mediastinum. Every lymph node was divided into two parts of the same size. One half part of the lymph node was examined by hematoxylin eosin (H&E) staining. If HE staining discovered metastasis, further examination was not needed. If HE staining failed to discover metastasis, then all the remaining lymph node samples of each patient were mixed together to undergo RT-PCR for cytokeratin 19 (CK(19)), a tissue marker of epithelium and epithelial tumors. RESULTS: (1) 195 lymph nodes from 25 patients with NSCLC were examined by H&E staining. 30 lymph nodes in 9 patients showed gross nodal metastasis and none showed micrometastatic tumor cells. (2) Of the 39 groups of mixed regional lymph node samples which were diagnosed to be devoid of metastases by H&E staining, 11 groups were found to have positive reactions to CK(19) mRNA. (3) Six of the sixteen patients staged as PN(0) had hilum lymph nodal micrometastasis, while 5 of nine patients with stage PN(1) had mediastinal lymph nodal micrometastasis (chi(2) = 54.063, P = 0.0043). CONCLUSION: (1) H&E staining can accurately detect gross nodal metastasis in the lymph nodes of the patients with NSCLC, but is unfit for detecting lymph nodal micrometastasis. (2) RT-PCR can facilitate the detection of occult micrometastatic tumor cells in the lymph nodes of NSCLC, and its assessment of nodal micrometastasis can provide a refinement of molecular stage for partial patients with stage I to II.

[Effect of MicroRNA-146b-5p on Adipogenesis of Mouse Bone Essence Derived Mesenchymal Stem Cells].

OBJECTIVE: To explore the effect of MicroRNA-146b-5p (miR-146b-5p) on the mouse bone essence derived MSC adipogenic differentiation. METHODS: MSC were isolated from bone essence of C57BL/6 mice. The expression level of miR-146b-5p in the process of adipogenic differentiation of MSC was detected by q-PCR; the role of miR-146b-5p mimics or inhibitors in the process of mouse bone essence derived MSC adipogenic differentiation was analyzed through oil red staining the expression of C/EBPα and PPARγ after cultured for 14 days was detected by q-PCR; the protein level of PPARγ after miR-146b-5p transfection was detected by Western blot. RESULTS: The MSC were successfully isolated from bone essence of mice, the q-PCR results showed an increasing expression level of miR-146-5p in the process of MSC adipogenic differentiation. Compared with the control group, MSC transfected with miR-146b-5p mimic could up-regulate the expression of miR-146b-5p (P<0.001), while miR-146b-5p inhibitor transfection could down-regulate the endogenous miR-146b-5p expression (P<0.01). After culture for 14 d, the result of Oil red staining showed that the miR-146b-5p inhibitor could inhibit adipogenic differentiation, while the miR-146b-5p mimic could promote the adipogenic differentiation of MSC. After induction for 14 d, compared with control, the PPARγ and C/EBPα in mimic group were higher expressed PPARγ and C/EBPα (P<0.01). Compared with induced group, the PPAPγ and C/EBPα were lower expressed in inhibitor group (P<0.05). The results of Western blot showed that the expression level of PPARγ was high in minic group, and it was low in inhibitor group. CONCLUSION: miR-146b-5p is up-regulated in the process of MSC adipogenic differentiation, and it promotes the adipogenesis of MSC originated from mouse bone essence.

[Preparation of Platelet-Rich Plasma from the White Slurry and Its Effect on MSC Proliferation].

OBJECTIVE: To isolate platelet-rich plasma(PRP) from the white slurry(WS), a depleted fraction of the clinical blood supply, so as to provide an easier method to harvest PRP for related studies and clinical use. METHODS: The protocols preparing PRP from whole blood and WS were compared. The morphological characteristics of the different PRPs were observed under transmission electron microscope; the expression of the platelet markers CD41a and CD42b were detected by the flow cytometry. Moreover, the ingredients of the PRPs were measured by using cytoanalyzer. for detecting the physiological function of the PRP, the harvested PRP were added to MSC culture and the cell proliferation was detected by using CCK-8 method. RESULTS: a large amount of PRP from WS was easier harvested. the WS-derived PRP shared similar morphological characteristics and ingredients as compared with whole blood-derived PRP. Importantly, the WS-derived PRP exhibited a higher expression of CD41a and CD42b than that of traditional PRP, which indicate that the WS is a promising reservoir for PRP. CONCLUSION: The WS can be used to prepare PRP, and the novel PRP share similar biological characteristics as traditional PRP prepared from whole blood. The present study provides an easier and economical method to harvest PRP and this findings may be helpful for PRP related studies.

[Establishment of mouse stably knockout of MYSM1 in MSC cell line C3H10T1/2 and its effect on the immune regulation in vitro].

OBJECTIVE: To aimed at the establishment of mouse stably knockout of MYSM1 mesenchymal stem cell(MSC) line C3H10T1/2, and to investigate its immunological capacity of MSC in vitro. METHODS: To establish the stably transfected MSC cell line by using CRISPR-Cas9 technology. Then the Flow cytometry, quantitative PCR and Western blot were employed to detect whether the MYSM1 have been knockout yet. Furthermore, the immune modulatory effect of MYSM1-/- MSC was tested by addition of MYSM1-/- MSC supernatant into spleen lymphocyte and Foxp3 culture. The mRNA expression of inflammatory cytokines such as interleukin-4, interferon-γ and interleukin-17 were detected by quatitatine PCR. RESULTS: The expression of MYSM1 was steadily knock out in MSC. In addition, MYSM1-/- MSC showed a stronger inhibitory effect on the expression of inflammatory cytokines. Therefore, the MYSM1 has been stably knocked out in C3H10T1/2. CONCLUSION: The mouse stably knockout of MYSM1 mesenchymal stem cells has been successfully established, the knock-out of MYSM1 in MSC can induce more potent immunosuppressive effects on cellular immune reaction in vitro. Our data laid a foundation for the further MSC-based applications in immune related diseases.

[Effects of Shock Wave on the Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells].

OBJECTIVE: To explore the effects of the shock wave on the capacity of mesenchymal stem cells(MSCs) to proliferate and differentiate into osteoblasts. METHODS: MSCs were isolated from the bone marrow of healthy donors. The human bone marrow MSCs(BM-MSCs) were divided into 3 groups including blank control group,osteoinduced group and shock wave group. The MSCs in blank control group were cultured with common mediam; the MSCs in osteoinduced group were treated with osteogenic agents and cultured; the MSCs in shock wave group were cultured with common medium and stimulated by shock wave. The morphology of MSCs in each groups were observed by micoscopy; the CCK-8 was used to detect the proliferation ability of MSCs; the alkaline phosphatase staining and von Kossa staining were used to evaluale the differentiation potential of MSCs in each groups. RESULTS: The results of CCK-8 revealed the shock wave could promote cell proliferation as compared with blank control group. The results of alkaline phosphatase and Von Kossa staining showed that the shock wave displayed a stronger ability to promote the human BMMSC differentiation into osteoblasts cells in comparison with the osteoinduced group. The blank control group was weakly positively stainined. CONCLUSION: The shock wave treatment can promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

[Modulatory Effect of Mouse Compact Bone-derived Suspending MSC on T Cells and It's Related Mechanisms].

OBJECTIVE: To investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms. METHODS: The suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected. RESULTS: The suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor ß1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A. CONCLUSION: The suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

[Effect of Knockdown of Vascular Cell Adhesion Molecule-1 on the Immunologic Regulation Capacity of Murine Mesenchymal Stem Cells].

OBJECTIVE: To establish the stably lower expression of vascular cell adhesion molecule-1 (VCAM-1) in MSC cell line (C3H10T1/2) by siRNA technology, and explore the effect of knockdown of VCAM-1 on the immunologic regulation capacity of murine MSC. METHODS: The mouse GV118-VCAM-1-RNAi retrovirus vector was constructed by gene recombination technology. The recombinant plasmid was identified by restriction analysis and sequencing, and then the recombinant plasmid GV118-VCAM-1-RNAi was transfected into 293 cells by Lipofectamine, and the supernatant was collected to transfect C3H10T1/2. Moreover, the VCAM-1 lower expression on MSC was evaluated by flow cytometry and fluorescent microscopy. The knockdown VCAM-1 MSC was sorted by flow cytometry. Furthermore, the inhibitory effect of the knockdown VCAM-1 MSC on lymphocyte proliferation was tested by lymphoblast transformation assay (LTT) and mixed lymphocyte reaction assay(MLR). RESULTS: The recombinant retroviral vector of knockdown VCAM-1 (GV118-VCAM-1-RNAi) was successfully constructed and transfected into mouse MSC cell line C3H10T1/2. The knockdown VCAM-1/MSC was obtained by flow cytometric sorting. The LTT and MLR assay showed that the immunosuppressive effect of MSC lower-expressing VCAM-1 dramatically decreased (P<0.05). CONCLUSION: Knockdown VCAM-1 in MSC can significantly down-regulate the inhibitory capability of MSC on the proliferation of T-cells. The data of this study laid an experimental foundation for studying effect of VCAM-1 transfecting into MSC on immune function.

[Effect of Vascular Cell Adhesion Molecule -1 Overexpression on Adipogenic Differentiation of Murine Mesenchymal Stem Cells and Its Mechanism].

OBJECTIVE: To investigate the effect of vascular cell adhesion molecule-1 (VCAM-1) gene overexpression on adipogenic differentiation of mouse mesenchymal stem cells(MSC) and explore its molecular mechanism. METHODS: VCAM-1 overexpression MSC (MIGR1-VCAM-1/MSC) and the empty plasmid transfection MSC (MIGR1/MSC) were induced to adipogenic differentiation, oil-red-O staining and real-time PCR were used to detect the adipogenic differentiation ability and the mRNA expression level of key transcription factors C/EBP α and PPAR γ. The activation of P38, ERK and JNK pathways were analyzed by Western blot. Furthermore, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the induced culture system and the alteration of the MSC adipogenic differentiation ability were evaluated. RESULTS: no matter in self or induced differentiation groups, the lipid droplets in MIGR1-VCAM-1/MSC became larger, the amount of adipocyte increased than that in MIGR1/MSC (P<0.01), the mRNA expression level of C/EBPα and PPARγ were upregulated, and JNK pathway were down-regulated while the P38 and ERK pathway were significantly up-regulated. The inhibition of JNK pathway of MIGR1-VCAM-1/MSC could lead to increased mRNA expression level of C/EBP α and PPAR γ, the amount of adipocytes increased (P<0.01), however, the inhibition of the P38 and ERK pathway of MIGR1-VCAM-1/MSC could lead to decreased mRNA expression level of C/EBP α and PPAR γ, and the lipid droplets and the number of adipocytes became smaller and less. CONCLUSION: The overexpression of VCAM-1 may promote MSC to differentiate into adipocytes through inhibiting JNK signaling pathway, activating P38 and ERK pathways.

Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function.

The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair.

[Isolation and Biological Characteristics of Rabbit Bone Marrow Plug-derived Mesenchymal Stem Cells].

OBJECTIVE: Though the rabbit is one of most widely used experimental animals for medical regenerative research, it remains difficult to culture mesenchymal stem cells (MSC) on a in large scale due to the extremely lower number and hematopoietic cell contamination. This study was aimed to establish a novel protocol to generate rabbit MSC by culturing bone marrow plugs instead of bone marrow cells so as to obtain a large amount of MSC with higher proliferation and self-renewal properties. METHODS: The primary MSC were generated from collagenase digested bone marrow plugs and bone marrow cells, respectively. The surface antigen profile of MSC was analyzed with flow cytometry and the cells were induced to differentiate into osteoblasts and adipocytes. The proliferation capacity of MSC were assessed by CCK-8 method. To test their self-renewal property, the colony forming unit-fibroblast assay was performed. Moreover, the cell yields of passage 1, 2, 3 and 4 were calculated. RESULTS: The bone marrow plug-derived MSC shared the typical fibroblast-like morphology same as bone marrow cells derived MSC. Moreover, the ratio of CD45 positive hematopoietic cells in bone marrow plug-derived MSCs was significantly lower than that of bone marrow cell-derived MSC. The results of multi-differentiation experiments showed that bone marrow-plug-derived MSC exhibited similar multi-potent property to their bone marrow counterparts. In addition, the results of CCK-8 and CFU-F assay demonstrated that bone marrow plug-derived MSC grew more robustly and more CFU-F were formed in the culture plates, which indicated that the cells possessed higher proliferation and self-renewal capacities. Promisingly, a larger amount of cells were harvested via using the new protocol. CONCLUSION: The purity and yields of the bone marrow plug-derived MSC are satisfactory compared with previous rabbit MSC isolation methods. The findings may be helpful for the research of regenerative medicine.

[Influence of donor mouse age on the establishment of murine acute graft versus host disease model after allogenic hematopoietic stem cell transplantation].

This study was purposed to elucidate the influence of donor mouse age on the establishment of murine acute graft versus host disease (aGVHD) model after allogenic hematopoietic stem cell transplantation. The male mice with 2-week-old, 10-week-old and 18-week-old mice (BALB/cH-2Kb) were taken as donors. The 8-week-old mice (BALB/c, H-2Kd) were selected as recipients. Each group animals were irradiated with 7.5 Gy (60)Co for total body, the recipient mice were injected intravenously with 1 × 107 bone marrow cells and 1 × 107 spleenoctyes from various donors in 4-5 hours after irradiation. Mouse transplant characteristics and survival were observed every day. The white blood cell number in peripheral blood of each group were counted at day 5, 10, 15, 20, 25 and 30 after transplantation. Furthermore, the pathological damage in the liver, spleen, lung and intestines were evaluated by sectioning and in situ hematoxylin-eosin (HE) staining. The results showed that compared with the 2-week-old and 10-week-old donor groups, mice received bone marrow (BM) cells and splenocytes from 18-week-old mice showed higher incidence of aGVHD, lower clinical GVHD scores and suffered from diarrhea, ruffled hair, a hunched posture, and diminished body weight. In contrast, mice received BM cells and splenocytes from 2-week-old donor mice indicated attenuated GVHD symptoms and survived longer. The histo-pathological analysis in 18-week-old donor group demonstrated the most serious pathological damage in the liver, spleen, lung and intestines. It is concluded that the donor age has been confired to have an obvious influence on the establishment of murine aGVHD model. This study lay an important foundation for establishing animal models and may be helpful for further study.

[Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.

[ICAM-1 regulates differentiation of MSC to adipocytes via activating MAPK pathway].

This study was aimed to explore the molecular mechanism of the regulatory effects of ICAM-1 on the differentiation of mesenchymal stem cells (MSC) to adipocytes. The murine MSC cell line C3H10T 1/2 was treated with the supernatants contained plasmid MIGR1-ICAM-1 and MIGR1-ICAM-1/MSC (high expression of ICAM-1), the activation of the pathway was detected by Western blot. The ICAM-1 modified MSC and its control cells named MIGR1/MSC were cultured in adipocyte medium with or without the inhibitors of the ERK, P38, and JNK pathway. Oil-red-O staining was used to detect the lipid accumulation, and the expression of C/EBPα and PPARγ in differentiation of MSC to adipocyte were examined by real-time-PCR. The results showed that the overexpression of ICAM-1 stably activated the ERK, P38, and JNK pathway in MSC. Inhibiting of the activation of ERK pathways by chemical inhibitors up-regulated the mRNA expression level of C/EBPα and PPARγ in MIGR1-ICAM-1/MSC while inhibiting of P38 pathway resulted in lower mRNA expression of the transcription factors. Consistent with the mRNA expression, the lipid droplets were getting smaller and number of adipocytes increased when P38 pathway was inhibited, while bigger lipid droplet and increased quantity of adipocytes were identified in MIGR1-ICAM-1/MSC with the addition of ERK pathway inhibitor. It is concluded that ICAM-1 may suppress MSC differentiate into adipocyte via activating ERK pathway, while it can maintain the adipogenesis of MSC though P38 pathway.

[Effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation].

This study was aimed to investigate the effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation. The male mouse BALB/c(H)-2d was totally irradiated with different radiation dose of (60)Co (TBI), then was infused with the same number of splenocytes from MHC matched DBA/2 male mice. After transplantation, the bodyweight, general appearance, hair changes, survival time and pathological damage were observed. The results indicated that compared to the control group (0 Gy) and the 7.0 Gy group, the mice irradiated with 7.5 Gy and 8.0 Gy showed cGVHD symptoms and obvious pathological damage. At the end of experiments (60 d after transplantation), all mice irradiated by 7.5 Gy survived while only 60% animals survived in the 8.0 Gy group. It is concluded that under infusion of 10(8) MHC matched splenocytes per mouse, 7.5 Gy irradiation is appropriate to efficiently establish cGVHD model. This study laid an important foundation for further studying the pathogenesis, biological characteristics, and intervention factors of cGVHD.