| ObjectiveNon-invasive micro-test technique (NMT), a selective micro electrode technology, which may obtain the multi-dimensional information of all kinds of ion or molecules in and out samples under a circumstance of not damaging the in vivo samples, can measure the in vivo material ion and small molecular flow velocity under real-time selectivity. This technology has advantages of non-invasive, long-time, multi-electrode, high resolution, high sensitivity, multi-angle measuring, etc, and may acquire the physiological characteristics and vital activity law that other techniques are hardly measuring. Therefore, a substantial breakthrough has been made in the fields of theoretical research and applications, so we can perform the real-time and dynamic measurements and studies to in vivo materials. In the past, NMT was mainly used in botanical physiology study, while it was further used in human cellular physiology in this trial. We have planned to adopt the NMT to conduct the real-time measurement for ion and small molecular flow velocity in and out a single oral squamous cancer cell under the conditions of not touching cells, not interfering the cellular activities, and not affecting internal and external cellular environment stability and regulatory mechanism in order to obtain the dynamic information of O2 and Ca2+ flow transmembrane alteration at tumor cell apoptosis. We have planned to adopt the NMT to investigate the transmembrane alteration of key molecular O2 and ion Ca2+ flow in a human oral squamous carcinoma cell after Rose Bengal mediated Photodynamic therapy (RB-PDT), and we have looked into and understood the mechanism of the diagnosis and treatment of oral squamous cell carcinoma using Rose Bengal in order to evaluate its effect. Meanwhile, it is aimed at provides a basis for further application prospects of NMT in the medical cytobiology.Methods1 Non-invasive micro-test technique (NMT) of real-time dynamic observation and recording is used for O2 and Ca2+ transmembrane flow of single living oral squamous carcinoma cell Ca127.2 The cytotoxicity of Rose Bengal in different levels to oral squamous carcinoma cell Ca127 was tested by MTT method, as well as the cytotoxicity of different levels of Rose Bengal-mediated PDT to oral squamous carcinoma cell Ca127. Moreover, under the conditions of same level and same excitation light source, the suppression function of Rose Bengal (10?M)-mediated PDT to proliferation of oral squamous carcinoma cell Ca127 was also tested.3 NMT real-time dynamic testing was performed on O2 and Ca2+ flow velocity alteration at Rose Bengal-mediated PDT-induced single oral squamous carcinoma cell apoptosis, as well as a study on signal pathway to response the PDT-induced single oral squamous carcinoma cell apoptosis.(1) The generation of Rose Bengal-mediated PDT-excited reactive oxygen species (ROS) was tested by fluorescent probe DCFH-DA.(2) The alternation of Rose Bengal-mediated PDT-induced cellular mitochondrial membrane potential (MMP) was tested by fluorescent probe Rhodamine 123.(3) Rose Bengal-mediated PDT-induced cellular apoptosis rate was tested by Annexin V-FITC/PI double-staining.(4) The expression levels of Rose Bengal-mediated PDT-induced intracellular cytochrome C, Caspase-3, Caspase-9 and PARP proteins were tested by Western blotting.(5) The real-time dynamic alteration of O2 and Ca2+ transmembrane flow at Rose Bengal-mediated PDT-induced single oral squamous cancer cell apoptosis was tested by NMT.Results1 O2 and Ca2+ transmembrane flow of a single oral squamous carcinoma cell Ca127 was tested by Non-invasive micro-test technique (NMT) real-time dynamic detection. The results suggested that the O2 in the Ca127 cell had the transmembrane inflow within 24 hours of experimental observation. The movement speed of molecular flow was fluctuated between 16.26 pmole/cm2.s and 23.99 pmole/cm2.s. However, the dynamics characteristics of transmembrane transport of Ca2+were quite different. The direction of ion flow was fluctuated, the transmembrane flow can be either inwards or outwards, of which the maximum movement speed of outflow was 5.84 pmole/cm2.s and 3.22 pmole/cm2.s for inflow.2 Experimental results revealed that Rose Bengal could be taken in by the oral squamous carcinoma cell Ca127 and entered into the cell nucleus and cytoplasm. Low concentration of Rose Bengal had no cytotoxicity to oral squamous carcinoma cell Cal27 whereas the low concentration of Rose Bengal-mediated PDT had significant cytotoxicity to oral squamous carcinoma cell Ca127, as well as significant inhibiting effects to cellular proliferation.3 Rose Bengal-mediated PDT led to the oral squamous carcinoma cell Ca127 intracellularly releasing a large number of ROS. The cellular ROS levels were significantly increased and 2.46-fold higher than that of non-treatment group. Intracellularly generated ROS levels showed a peak 2 hours after RB-PDT treatment and 2.84-fold higher than that of non-treatment group. ROS release led to a decrease of intracellular MMP, only 39.21% at end of 24 hours of observation. Meanwhile, it induced the successive occurrence of oral squamous carcinoma cell apoptosis. The apoptosis rate appeared a peak 2 hours after RB-PDT treatment and reached by 18.55%. Then it was gradually going up and reached up to 30.57% 24 hours after RB-PDT treatment. Western blotting test results showed that the intracellular cytochrome C immediately released the protein expression after being excited by PDT. A high protein expression in Caspase-3, Caspase-9 and PARP was turned up 2 hours within the observation period.4 O2 and Ca+ transmembrane flow velocity changes at Rose Bengal-mediated PDT-induced single oral squamous carcinoma cell Ca127 apoptosis was tested by NMT real-time dynamic detection. Compared with the untreated group, RB group and PDT+NAC group, O2 intramolecular flow speed was decreased in the early stage (2 hours prior to the observation) of real-time dynamic detection. O2 movement speed (6.43 pmole/cm.s) was minimum at 2-hour time point and O2 intramolecular flow speed was increased in the later stage. However, the movement rate of Ca2+ ion flow was significantly higher than that of the untreated group, RB group and PDT+NAC group, either inflow or outflow. The movement rate of Ca2+ ion flow speed (9.53 pmole/cm.s) at 2-hour time point reached a peak where Ca2+ ion flow was turning into the inflow from the outflow. This result is consistent with above-mentioned results of apoptosis rate by V-FITC/PI double staining test and protein expression by Western blotting. Two-hour is a key observing time point worth to focus on.Conclusion1 Non-invasive micro-test technique can be adapted in medical cytobiology, which is a excellent method to study cellular pathophysiology under the conditions of not touching cells, not interfering the cellular activities, and not affecting internal and external cellular environment stability and regulatory mechanism.And NMT have great application prospect in the study of physiological characteristics and regulation of cellular activities.2 This study has successfully tested the O2 and Ca2+ transmembrane flow of single oral squamous cancer cell Ca127 during the normal growth metabolic process using NMT real-time dynamic detection, which provide important scientific foundation for follow-up research.3 This study has also investigated Rose Bengal photosensitizer-induced oral squamous cancer cell apoptosis and PDT killing effects to cells. The results suggested that mass ROS was released in the cell Ca127 and the mitochondrial cell membrane was damaged after Rose Bengal light-dynamic effects thereby lowering the MMT, damaging the cells, releasing the cytochrome C, and activating the Caspase reaction. The intracellular cytochrome C, Caspase-3, Caspase-9 and PARP protein expression levels have been rising. Therefore, we speculated that the alteration of 02 and Ca2+ responded to mitochondrion dependent approach-induced apoptosis signals after PDT effects, which can be used as indicators of tumor cell apoptosis. |
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