The Proliferation And Differentiation Of The Intestine Stem Cell In Silkworm, Bombyx Mori

Stem cell is a hot topic of research in biological research, they are defined by virtue of their functional speciality including proliferation, self-renewal, and differentiation. The intestinal tract has a rapid epithelial cell turnover, the process is regulated and maintained by a subpopulation of stem cells, which could proliferate and differentiate into all types of intestinal epithelial cells.Silkworm (Bombyx mori) is a representative of lepidopteran insects and a domesticated holometabolous insect, and its midgut epithelium is renewed during each molting and replaced during metamorphosis. However, the research about intestinal stem cell in silkworm remains rare, the exact identity of the characteristic about stem cells and the regulation of stem cell proliferation in response to environmental challenge are still largely unknown.To better address this question, we examined the morphological changes of silkworm midgut along with the growth, development and metamorphosis. Used bromodeoxyuridine5-Bromo-2-deoxyUridine (BrdU) and Phospho-Histone H3antibody (PHH3) as proliferation markers, we found that some small midgut cells located near the basement membrane in silkworm are either BrdU-positive or PHH3-positive. The main results are as following:1Histological observation of silkworm midgut at larval stageThe midguts from different stage of silkworm are fixed in Bouin’s buffer and embedded in paraffin. The morphological changes of the midgut structure during development and metamorphosis in silkworm(bombyx mori) were traced by H.E staining. The midgut structure of silkworm during larvae instar is a pseudostratified epithelium. We have determined three types of cells including columnar cells (CCs), goblet cells (GCs) and intestinal stem cells (ISCs) in epithelial lamina. GCs present a large pear-shaped cavity and has a basally located nucleus, whereas the CCs with a central nucleus and characterized by a thick brush border toward the lumen, and small round cells located near the basement membrane increased before each moulting.We also analyse that the number and the size of midgut increased before each moulting stage, then went back to the original state as displayed in the next instar. The result showed that the total number of cells increased along with the growth and development, which is confirmed by DAPI staining to visualize the nuclei. And the small round cells also increased and displayed a higher percentage before each moulting.2Immunofluorescence of midgutISCs were scattered along the basement membrane, we have used combinatorial approaches to characterize the midgut remodeling of silkworm with an emphasis on proliferation of midgut cells. A few of cells were labeled by bromodeoxyuridine5-Bromo-2-deoxyUridine (BrdU) and Phospho-Histone H3antibody (PHH3), and most of small BrdU-positive midgut cells located near the basement membrane in silkworm and co-localiaed with PHH3.3BrdU label retentionMoreover, after injections of BrdU (15ug) every eight hours for three consecutive times, we discovered that a distinct population of cells displaying BrdU-positive label-retaining cells (LRCs). We found that～46.8%of BrdU-positive cells at24hours after the final BrdU injection in silkworm midgut. Thereafter, the BrdU-positive population is gradually decreased followed the time course, it’s only reach to～0.08%at144hours.