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| A Simple and Efficient In Vitro Isolation and Culture System for Bone Marrow-Derived Precursor Cells (DC) in Rats |
| Huang Jiang-bo,LUO Zhi-gang,LIU Yu-ming,et al |
| Department of Urology, the Second Hospital Affiliated to Nanhua University, Hengyang, Hunan 421001, China |
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Abstract 【Objective】To establish a simple and efficient in vitro isolation and culture system for bone marrow-derived precursor cells (DC) in rats.【Methods】 Granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin -4 (IL-4) were used to induce and separate rat bone marrow hematopoietic progenitor cells to get immature dendritic cells after 7 d (imDC), immunomagnetic flow cytometry was used before and after purification of the surface of imDC to test positive expression rate of OX62. Lipopolysaccharide (LPS) lasted for 2 D to stimulate imDC maturation to get mature dendritic cells (mature dendritic cell, mDC), flow cytometry was used to detect imDC and mDC cell surface CD86, CD80, RT1B positive expression rate ; ELISA was used to detect the secretion of IL-12 in vitro in two groups of cells, mixed lymphocyte reaction (MLR) was used to determine lymphocyte proliferation ability of cells in two groups.【Results】Purified imDC surface OX62 positive expression rate was 91.9%, significantly higher than that before purification 82.8% (P<0.05); imDC, CD80, CD86 on the surface of the positive expression rates of RT1B were 11.48%, 6.25%, 77.2%, stimulated by LPS after the maturity of the mDC surface CD86, CD80 and RT1B positive expression rates were 91.38%, 93.54%, 98.6% imDC; cell IL-12 secretion level (36±9) pg/mL, was significantly lower than that of mDC (228 ±27) pg/mL (P<0.01) MLR results in mDC group; ability to stimulate T lymphocyte proliferation was significantly higher than that of the imDC group.【Conclusion】In this experiment, the cultured cells were identified by three aspects (cell morphology observation, surface molecular markers and functional status), and it was proved that the combination of GM-CSF+IL-4 could induce high purity and large numbers of DC in vitro; through the purification of immunomagnetic beads, a relatively large number of DC with high purity can be obtained to meet the experimental requirements.
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Received: 06 March 2017
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2018, Vol. 35 
