| Objective: Investigated whether ultrasound combined with BCNU-loaded lipid contrast agents(BLCA)could induce apoptosis in C6 cells and inhibit growth in C6 gliomas implanted transcranially into Wistar rats. Discussing the effects on therapy of ultrasound combined with BLCA in vitro and vivo, and treatment mechanism about it , to support experimental foundation for clinical applications.Methods:1. Prepare cammustine lipid ultrasound contrast agent and study its characterizations: Preparing BLCA with mechanical oscillation. Density was detected and analyzed with microscope. Malvern determine intensity, disposition and Zeta potential. RP-HPLC detect envelopment rate. Cryodesiccated-microbubbles were sterilized by 60Co irradiation. Following injected it through rabbit ear vein, effects of ultrasounic enhanced imaging in rabbit liver was observed.2. Apoptosis effects of ultrasound with BCNU-loaded lipid microbubbles on C6 cells in vitro: The OD of C6 cells in different groups points were detected with MTT assay at different time, the inhibitory rates of C6 cells were calculated, and required parameters was determinated. Apoptosis rates(FITC-Annexin V/PI double staining)and cell cycle (PI staining)in C6 cells of different groups was determined. After AO-EB double staining C6 cells, Observed morphological changes of C6 cells with fluorescence microscope. The C6 cells'ultrastructure changes were observed in TEM and SEM.3. Ultrasound with BCNU-loaded lipid microbubble to inhibit rat intracranial glioma in vivo: C6 cells were injected into the right caudate nucleus of Waster mice with a guide-screw system. Analyzing MRI head imaging and histological features to determine whether models were successful developed. Forty rats with C6 gliomas were randomly divided into four groups:①contrast group,②BCNU alone group,③ultrasound alone group,④ultrasound+BLCA group. Tumor volume of model rats was observed by MRI on 7 d,14 d and 21d. RT-PCR semi-quantitative detected expression of BAX, BCL-2 in C6 gliomas.Results:1. The physical characteristics of BCNU-loaded lipid contrast agents: The density of microbubble was (2.6~3.1)×109/ml. The intensity mean was (1.05±0.20)μm. The potential mean is (-12.93±1.84)mV. The envelopment rate mean was (42.6±0.11) %. After injecting this microbubble, good and sustained ultrasounic enhanced images in rabbit liver were seen.2. Ultrasound with BCNU-loaded lipid microbubbles induced apoptosis in C6 cells: At the time point of 6 h, compared with other groups , C6 cells'proliferation were obviously inhibited and its apoptosis rate significantly increased in ultrasound+ BLCA group(P<0.01). Ultrasound-induced BLCA destruction could reduce the ratio of S-phase in C6 cells. The apoptosis morphological change in C6 cells was observed with fluorescence microscope and electron microscope.3. The animal experiments of ultrasound with BCNU-loaded lipid microbubbles inhibited rat glioma growth: The rat intracranial glioma model was developed successfully. MRI showed that tumor volume in ultrasound+BLCA group is smaller than other groups, and there was a significant difference (P<0.01). Ultrasound with BLCA could decrease the expression of Bcl-2(anti-apoptosis gene) and Bcl-2/Bax ratio (P<0.01), but there is no change in Bax (apoptosis gene).Conclusions:1.The BLCA was constructed successfully. It had small size, distributed evenly, good ultrasounic enhanced-imaging in rabbit liver, and in line with the general characteristics of microbubbles.2. Ultrasound with BLCA could induce apoptosis in C6 cells and against C6 gliomas implanted transcranially into Wistar rats. Ultrasound with BLCA may inhibit the rat gliomas growth though decreasing the expression of Bcl-2(anti-apoptotic gene) and Bcl-2/Bax ratio. The study offered a new way to the treatment of gliomas. |
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