OBJECTIVEThis study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease.

METHODSBone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates.

RESULTSFlow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day.

CONCLUSIONThe whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

Objective:This paper aims to clone the cDNA sequence of limonene-3-hydroxylase（StL3OH） in Schizonepeta tenuifolia and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of StL3OH gene screened from transcriptome sequencing data of S. tenuifolia and the cDNA sequence of StL3OH gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The StL3OH gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that StL3OH gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of StL3OH gene. Conclusion:The cDNA sequence of StL3OH gene was cloned from S. tenuifolia for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of StL3OH gene in the accumulation and biosynthesis of monoterpenes in S. tenuifolia.

OBJECTIVETo investigate the common mutation spectrum of α- and β-thalassemia in Yunnan childbearing-aged population.

METHODSThe common mutation types of α- or β-globin genes were detected by multiple Gap-PCR and the PCR-reversed dot blotting, and the unknown mutation types were determined by DNA sequencing in DNA samples of hypochromic microcytic anemia patients and carriers who were confirmed to be positive by serologic screaning, then the mutation types of globin in Yunnan population were analyzed statistically.

RESULTSA total of 40 kinds of mutation types were detected in 685 detected persons, among them the 3 commonest mutation types of α-globin genes were --(SEA)/αα (49.09%), -α(3.7)/αα (36.67%) and α(CS)α/αα (8.79%), the 3 commonest genetypes of β-globin gene were CD26(GAG>AAG)/N (43.78%), CD41-42(-CTTT)/N (20.1%) and CD17(AAG>TAG)/N (18.9%). There were 348 Han and 212 Dai ethnic persons in 685 cases, but their mutation of globin genes were different between these 2 ethnic groups. The results also showed that the gene mutation types were mostly concentrated in Dai ethnic individuals, since 28 of 38 detected α-β-thalassemia cases were Dai ethnic individuals.

CONCLUSIONThe mutation spectrums of α- and β-globin genes in Yunnan childbearing-aged population are diverse and different from that in other areas of China.

Alpha-Globulins ; genetics ; Anemia, Hypochromic ; ethnology ; genetics ; Asian Continental Ancestry Group ; China ; DNA Mutational Analysis ; Ethnic Groups ; genetics ; Genetic Testing ; Heterozygote ; Humans ; Mutation ; Polymerase Chain Reaction ; alpha-Thalassemia ; ethnology ; genetics ; beta-Globins ; genetics ; beta-Thalassemia ; ethnology ; genetics