Chondrogenic differentiation of ChM-I gene transfected rat bone marrow-derived mesenchymal stem cells on 3-dimensional poly (L-lactic acid) scaffold for cartilage engineering.

We have explored the role of Chondromodulin-I (ChM-I) in chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) in 3-dimensional (3D) scaffold for cartilage tissue engineering. BMSCs of Sprague Dawley (SD) rats were cultured on poly-(L-lactic acid) [PLLA] scaffolds with different pore sizes (80-200 µm, 200-450 µm) with or without surface modification by chitosan. Cell viability, proliferation, and morphology were measured using confocal microscope and the CCK-8 method. Untransfected BMSCs, BMSCs expressing pcDNA3.1(+), BMSCs expressing plasmid pcDNA3.1 (+)/ChM-I were cultured on 3D scaffolds in standard growth medium or transforming growth factor-ß1 (TGF-ß1) supplemented chondrogenic induction medium in vitro for 3 weeks and the expression of collagen type II was determined. Cell-scaffolds constructs were implanted subcutaneously for 3 months in vivo. BMSCs had a higher viability and proliferation in PLLA scaffolds of pore size 200-450 µm than that of 80-200 µm, and surface modification with chitosan did not enhance cell attachment. The ChM-I gene enhanced chondrogenesis and increased collagen type II synthesis. Immunohistochemistry from in vivo study showed enhanced cartilage regeneration in BMSCs expressing pcDNA3.1 (+)/ChM-I on 3D PLLA scaffolds. It also demonstrated that TGF-ß1 might promote chondrogenesis of rat BMSCs by synergizing with the ChM-I gene. ChM-I could be beneficial to future applications in cartilage repair.

Development and validation of an LC-MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study.

Bullatine A is a diterpenoid alkaloid of Xue-Shang-Yi-Zhi-Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC-MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG-C18 column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple-reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32-440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection.

Proteomic analysis of chondromodulin-I-induced differentiation of mesenchymal stem cells into chondrocytes.

To identify novel proteins that might help clarify the molecular mechanisms underlying chondromodulin-I (ChM-I) induction of mesenchymal stem cells (MSCs) differentiate into chondrocytes. MSCs are triggered to differentiate into chondrocytes, which are recognized as important factors in cartilage tissue engineering. ChM-I is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. In this study, the proteomic approach was used to evaluate protein changes between undifferentiated MSCs and ChM-I-transfected MSCs. The expression of the protein spots was analyzed using two-dimensional gel electrophoresis. Then, 14 protein spots were identified between MSCs and ChM-I-transfected MSCs. 309 proteins were identified using mass spectrometry (MS). The differentially regulated proteins were categorized and annotated using Protein Analysis Through Evolutionary Relationships (PANTHER) analysis with the aid of the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. These proteins are included in a variety of metabolic pathways and signal transduction pathways, such as focal adhesion, glycolysis, actin cytoskeleton regulation, and ribosome. These results demonstrate novel information about the molecular mechanism by which ChM-I induce MSCs to differentiate into chondrocytes. These results also provide a solid foundation for the development of tissue-engineered cartilage.

Analysis of Active Components and Proteomics of Chinese Wild Rice (Zizania latifolia (Griseb) Turcz) and Indica Rice (Nagina22).

The ancient Chinese wild rice (Zizania latifolia (Griseb) Turcz) (CWR) has valuable biological and medicinal functions. To assess the advantages lost in modern cultivated rice after domestication, we compared the composition of bioactive compounds and the results of proteomic analysis with those of Indica rice (N22). We used routine methods to determine the protein, total dietary fiber, amino acid, mineral substance, plant secondary metabolites, and amino acid composition of CWR and N22. The protein and mineral contents of CWR were two times that of N22, and the levels of calcium, potassium, magnesium, chromium, iron, and zinc were significantly higher than those of N22 (P < .05). There was ∼7.6 times more dietary fiber in CWR than in N22, but fewer carbohydrates (P < .05). Anthocyanins and chlorophyll were detected in CWR, but were absent from N22. Compared with N22, CWR had 53, 19, and 5.4 times higher (P < .05) levels of saponins, flavonoids, and plant sterols, respectively. The amino acid score of CWR was 66.6, which was significantly higher than N22. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the main seed proteins of CWR were glutelins, including both acid and alkaline subunits, which were approximately twice those of N22. To investigate the differences in protein profiles between CWR and N22, we conducted two-dimensional electrophoresis (2-DE) analysis of the total proteins in the seeds of the two rice species. 2-DE gels revealed 19 differentially expressed proteins. Information obtained from peptide mass fingerprinting indicates that glutelin precursor caffeoyl coenzyme A (CoA) O-methyltransferase and putative bithoraxoid-like protein can provide good gene sources for improving rice quality.

Mesenchymal stem cells display different gene expression profiles compared to hyaline and elastic chondrocytes.

Cartilage has a poor intrinsic repair capacity, requiring surgical intervention to effect biological repair. Tissue engineering technologies or regenerative medicine strategies are currently being employed to address cartilage repair. Mesenchymal stem cells (MSCs) are considered to be an excellent cell source for this application. However, the different gene expression profiles between the MSCs and differentiated cartilage remain unclear. In this report, we first examined the gene expression profiles between the MSCs, hyaline and elastic chondrocytes, and then identify candidate genes, which may be important in the process of MSC differentiation into hyaline and elastic cartilage. Several hundred differentially expressed genes were screened initially by microarray, including 417 simultaneously up-regulated genes in both hyaline and elastic chondrocytes, with 313 down-regulated genes. Several genes were identified that were up-regulated in hyaline chondrocytes while down-regulated in elastic chondrocytes. Both RT-PCR and western blot analysis were consistent with those results obtained by microarray analysis. Chondromodulinl (Chm1) was found to be highly expressed in MSCs differentiating to hyaline and elastic cartilage. Both collagen type II, alpha 1 (Col2a1) and cartilage homeo protein 1 (Cart1) were also highly upregulated and may be important early differentiation of MSCs to hyaline cartilage.

[Effects of different vectors and gene fragments on antigen expression of hepatitis E virus DNA immunization].

AIM: To investigate the effects of different vectors and gene fragments on antigen expression of hepatitis E virus (HEV) DNA immunization. METHODS: Gene fragments encoding p166 and p179, which contain the neutralization antigenic epitopes of a Chinese strain of HEV genotype IV, were cloned into two different eukaryotic expression vectors (pTR421 and pCDNA3.1), respectively. The in vitro expression level of p166 and p179 in HepG2 cells transfected by each of the recombinant plasmids with lipofectamine2000 was examined by means of immunofluorescence and Western blot. Meanwhile, the in vivo expression level in muscles of mice was examined with immunohistochemistry staining. RESULTS: Four recombinant plasmids, pTR421-166, pTR421-179, pCDNA3.1-166 and pCDNA3.1-179, were constructed successfully and confirmed correct with restriction endonuclease analysis and nucleotide sequencing. The antigen expression was only detected in HepG2 cells transfected by pTR421-179 and in myocytes of the mice injected with pTR421-179. Neither in vitro nor in vivo antigen expression was detected with pTR421-166 although p166 was only 13 amino acids shorter than p179 at N terminus. Neither pCDNA3.1-166 nor pCDNA3.1-179 was expressed in vitro and in vivo. CONCLUSION: Selection of the vectors and gene fragments is critical to HEV gene expression and HEV DNA vaccine.