PGC-1α prevents apoptosis in adipose-derived stem cells by reducing reactive oxygen species production in a diabetic microenvironment.

AIMS: To examine whether overexpression of peroxisome proliferator activated receptor-gamma coactivator-1 alpha (PGC-1α) can prevent apoptosis in adipose-derived stem cells (ASCs) by reducing reactive oxygen species (ROS) production and enhancing mitochondrial function in a diabetic environment. METHODS: After the isolation, expansion and characterisation of rat ASCs, we overexpressed PGC-1α in ASCs using an adenoviral vector encoding green fluorescent protein (GFP) or PGC-1α and tested the apoptotic effect under conditions of high glucose, hypoxia and serum deprivation. The production of intracellular ROS and mitochondrial ROS was evaluated using dihydroethidium and CM-H2XRos fluorescent probes. RESULTS: Under conditions of high glucose, hypoxia and serum deprivation, the overexpression of PGC-1α in ASCs decreased apoptosis and led to an increased survival rate. The ASCs modified with PGC-1α produced lower intracellular and mitochondrial ROS. The mitochondrial morphology and structure in the PGC-1α-ASC group remained relatively complete compared with the control group. CONCLUSIONS: These results reveal a crucial protective role for PGC-1α in the treatment of diabetes mellitus and its complications using stem cells therapy.

Postnatal development of interstitial cells of Cajal in mouse colon in response to Kit signal blockade with Imatinib (Glivec).

This study investigated the response of interstitial cells of Cajal (ICC) in postnatal mouse colon to treatment with Imatinib (Glivec), a potent inhibitor of Kit receptor). ICC were revealed by immunofluorescent staining on frozen cross-sections and whole-mount preparations by anti-Kit and DOG1 antibodies. Kit and p-Kit protein were also evaluated by Western blot. After administration of Imatinib for 4 days beginning at 8 days post-partum (P8), the mean density of Kit+ ICC, which were localized around the myenteric nerve plexus (ICC-MY), within smooth muscle layers (ICC-IM) and in the connective tissue beneath the serosa (ICC-SS), was dramatically decreased to about 50% when compared with controls, but those Kit+ cells located at the submucosal border of circular smooth muscle layer (ICC-SM) seemed to be unchanged in both cell number and morphology. A small number of DOG1+/Kit(-) cells appeared during Imatinib administration. However, these Kit+ ICC were not changed in mice even after 12 days of Imatinib treatment from P24. When Imatinib was discontinued, the number of ICC recovered to normal within 4 days. Our results indicate that the postnatal development of ICC in the mouse colon is Kit dependent, but ICC-SM are unlikely, and the Kit dependence of ICC development is also age-dependent.

Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit(+) ICC, which situated around myenteric nerve plexus (ICC-MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC-IM) and in the connective tissue beneath serosa (ICC-SS) began to appear. ICC-SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased approximately 30 folds at P56 than that at P0. Some Kit(+)/Ki67(+) and Kit(+)/BrdU(+) cells were observed in ICC-MY, ICC-IM and ICC-SS, but not in ICC-SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.

[Inhibitory effect of short hairpin RNA on expression of Hax-1 gene in HeLa cells].

AIM: To construct the short hairpin RNA (shRNA) eukaryotic expression vector specific for HS1-associated protein X-1 (Hax-1) and investigate the inhibitory effect of it on Hax-1. METHODS: According to the design rules of shRNA, the specific sequence of 19 nucleotides were selected from Hax-1 cDNA sequence and designed as the cDNA template of siRNA. ShRNA vector pGenesil-Hax-1 was constructed by recombining the synthesized specific sequence with siRNA expression vector pGensil-1. After the recombinant plasmids were transfected into HeLa cells, RT-PCR technique and Western blot were applied to analyze mRNA and protein expression of Hax-1. RESULTS: The results of RT-PCR showed that the down-regulation of Hax-1 mRNA expression was found in the pGenesil-Hax-1 transfected group, but not in the pGenesil-1 transfected group or the negative control group (P<0.01). The expression of Hax-1 protein decreased by 70% in the pGenesil-Hax-1 transfected group compared with the negative control group. CONCLUSION: Hax-1 gene expression can be inhibited markedly by specific shRNA in HeLa cells, which establishes the experimental foundation for further study on the biological functions of Hax-1 in HeLa cells.