Versatile on-stage microfluidic system for long term cell culture, micromanipulation and time lapse assays.

We report here a versatile on-stage microfluidic cell culture and assay system which is compatible with different microscopes and sensors, can simultaneously perform steps of long term cell culture, high throughput time lapse cell assays/imaging, and cell micromanipulations. With the system, we cultured a variety of cells for different periods of time and monitored their cell morphology, migration and division. We also performed a series non-invasive real time in situ time lapse assays and micromanipulations on different cells. They include: the first time lapse imaging and measurements on the instantaneous variations of morphology, biomechanical properties and the intracellular protein of human red blood cells in responding to pH fluctuation, drug action and electromagnetic radiation; the first continuous time lapse Raman micro-spectroscopy on a CHO cell in different phases of its entire life cycles; the micro-transfection of GFP into B16 cells and the follow up observation of the cell's morphology and expressed GFP fluorescence varying with incubation time and cell generations. The performance of these experiments not only demonstrated the capability of the system, but also proposed a variety of novel methods for obtaining time- and spatially-resolved information about the cellular and molecular heterogeneity and transformation during development or stimulations.

Protective effect of non-mitogenic human acidic fibroblast growth factor on hepatocyte injury.

AIM: To study whether non-mitogenic human acidic fibroblast growth factor (nm-haFGF) has protective effects on H(2)O(2)-induced hepatocyte injury in vitro and CCl(4)-induced hepatocyte injury in vivo. METHODS: (i) HL-7702 hepatocytes were incubated with different concentrations of nm-haFGF for 12 h, and then the activity of lactate dehydrogenase (LDH) in culture medium was detected, and genomic DNA electrophoresis analysis was observed after being exposed to H(2)O(2) (8 mmol/L) for 4 h. Proximately, apoptotic rates and protein expressions of Bcl-2 and Bax of HL-7702 cell were detected after being exposed to H(2)O(2) (0.2 mmol/L) for 20 h. (ii) Being injected intraperitoneally with nm-haFGF, mice were treated with CCl(4) intraperitoneally to induce hepatic injury. Twenty-four hours later, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured and histopathologic changes were evaluated. RESULTS: (i) In vitro tests: LDH activities and apoptotic rates decreased, the protein expression of Bcl-2 increased and Baxdecreased in nm-haFGF-treated groups at the concentrations of 100 150 and 200 ng/mL, compared with that in the model control group, which was treated with H(2)O(2) alone. The genomic DNA remained nearly intact at the concentrations of 150 and 200 ng/mL. (ii) In vivo tests: serum ALT and AST in nm-haFGF-treated groups (10 mug/kg and 20 mug/kg) were much lower as compared to the model control group, which was treated with CCl(4) alone. Histological examination showed that nm-haFGF markedly ameliorated hepatocytes vacuolation, cloudy swelling and inflammatory cells infiltration induced by CCl(4). CONCLUSION: nm-haFGF had protective effects against H(2)O(2)-induced hepatocyte injury in vitro and CCl(4)-induced acute liver injury in vivo.

[Protective effect of non-mitogenic haFGF on retinal injury induced by N-methyl-N-nitrosourea in Sprague-Dawley rats].

AIM: To study the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on retinal injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats and its mechanism. METHODS: Female rats of 50-days-old were injected with MNU (60 mg x kg(-1)) intraperitoneally, and three doses of nm-haFGF (1.25 microg, 2.5 microg and 5 microg in one eye of each rat) were injected, separately, into vitreous body of one eye of each rat twice a day at 0 and 12 h after MNU treatment. 24 h later, apoptotic index of photoreceptor cells was detected by TUNEL labeling and the expressions of Bcl-2 and Bax were analyzed by Western blotting. At the 7th day, retinal injury was evaluated based on retinal thickness. RESULTS: Compared with model group, apoptotic index of photoreceptor cells was significantly reduced in nm-haFGF groups at the dose of 1.25 microg and 2.5 microg in one eye of each rat at 24 h, and the total retinal thickness as well as the outer retinal thickness markedly increased 7 days after MNU, respectively. The expressions of Bcl-2 increased and that of Bax decreased adversely after being injected with different doses of nm-haFGF. CONCLUSION: nm-haFGF partially suppressed retinal injury induced by MNU in Sprague-Dawley rats. The mechanism could be related to up-regulation of Bcl-2 and down-regulation of Bax.

[Instant effect of temperature on the oxygen carrying capacity of single living intact red blood cell].

The instant effect of temperature on the absorption spectra of the hemoglobin in single living intact red blood cells was investigated, by employing a highly sensitive fast multi-channel micro-spectrophotometer system to perform non-invasive, in situ, real time measurements on the cells. It was found that both the heights and position of the specific peaks in the absorption spectra of intercellular hemoglobin were changed with temperature, indicating that the oxygen carrying capacity of red blood cells varies with temperature. The correlations of the structure and concentration as well as the function of hemoglobin, and the molecular mechanism were also discussed.

Protective effects of mutant of acidic fibroblast growth factor against cerebral ischaemia-reperfusion injury in rats.

OBJECTIVE: To investigate the protective effect of a mutant of acidic fibroblast growth factor (MaFGF) against cerebral ischaemia-reperfusion injury in rats. METHODS: Sixty male Sprague-Dawley rats were randomly divided into six groups as follows: sham-operated group, untreated group, 20microg/kg, 40microg/kg and 80microg/kg MaFGF-treated groups and also the positive control group. Cerebral ischaemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. Different dose of MaFGF were infused intravenously at 1h after middle cerebral artery (MCA) occlusion. Nimodipine was used as positive control. The behaviour deficit score, brain-infarcted area, brain oedema degree, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected at 24h after reperfusion. RESULTS: The results showed that MaFGF at the dose of 20microg/kg, 40microg/kg and 80microg/kg significantly alleviated brain injury. Compared to untreated group, the behaviour deficits were much less severe, the brain oedema alleviated obviously, the MDA contents decreased and SOD activity increased dramatically in MaFGF-treated groups respectively. The efficacy of MaFGF was similar to that of nimodipine. CONCLUSION: The results demonstrate that MaFGF has neuroprotective effect against brain injury resulting from focal ischaemia-reperfusion in Sprague-Dawley rats.

Absorbable collagen sponge combined with recombinant human basic fibroblast growth factor promotes nerve regeneration in rat sciatic nerve.

Recombinant human basic fibroblast growth factor (rhbFGF) is a peptide with many bioactivities such as promoting proliferation and migration of various cells. It plays an important role in neuroprotection and enhancement of nerve regeneration. Due to its short half-life in the body, local administration by injection is limited. To prolong the bioactivity of rhbFGF and to enhance its biological effects, absorbable collagen sponge was used as matrixes and carriers for controlled release of rhbFGF. The effects of rhbFGF soaked into an absorbable collagen sponge (rhbFGF/ACS) for the repair of rat sciatic nerve injury were evaluated. The functional, electrophysiological and histological examinations demonstrate the treatment with rhbFGF/ACS can enhance rat sciatic nerve repair, and its effectiveness is better than free rhbFGF alone. It is concluded the rhbFGF/ACS is a promising biomaterial to improve the repair and regeneration of sciatic nerve injury.