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Induction Apoptosis in a short hair follicle RNA (shRNA) can be an effective and promising procedure for cancer treatment. Ultrasound-focused microbubble (DTM) is an excellent technique; however, there are limited resources available to demonstrate the potential and add guidance for this technology. The aim of this study was to improve this process and to explain the effects of genetic inhibition and the induction of apoptosis in vitro. Embryonic stem cells are collected and implanted. ShRNA vectors are enhanced, and the UTMD process is tested to determine whether it is suitable for transmitting shRNA to cells. Cells were then analyzed using flow cytometry. The results show that optimal radiation techniques allow for high transfer efficiency and do not affect the stability of plasmid DNA. We conclude that regeneration with shRNA expression trackers, introduced by UTMD high-level states, causes cell proliferation as a sign of cell proliferation, laying the foundation for further medical research. of cancer yii.
Apoptosis is an important physiological tool of the cell and is essential for tumor development and development. The restricted control of apoptosis maintains normal homeostasis with cells and morphogenesis of the human body. A clear strategy for treating cancer is to look at lesions that reduce apoptosis in tumor cells (1). Different molecular systems play a role in establishing apoptosis (2, 3). Survivin, the smallest member of the mammalian inhibitors of the apoptosis protein family (IAP), has been implicated in a number of mechanisms that protect cells from apoptosis (4). Excessive survival of cancer survives the apoptotic test site and prefers non-functional development with mitosis (5).
Cell Cycle Analysis By Flow Cytometry Propidium Iodide
Genetic modification by RNA (RNAi) interference using a small amount of RNA (siRNA) or producing stem-loop RNA [hairpin RNA (shRNA) short-hairpin or artificial microRNA (miRNA)] has been shown to work against le survivin in vitro le in vitro. vivo (6–8). However, due to its short and short life span of siRNA vivo transmission, the use of shRNA receptor receptors, under the guidance of RNA polymerase III manufacturers such as U6 and H1 , can be a powerful tool for the treatment of cancer. (9). , 10). shRNA is much more potent than siRNA in an intermediate attack, and the difference arising from the successful rendering of siRNA to cytosol is compared with the transfer of shRNA to the nucleus (11). In addition, shRNA is more efficient than synthetic miRNA which is much quieter, independent of ionization and testing system (12).
Cell Cycle Analysis With Propidium Iodide (pi). A Flow Cytometric…
However, the use of shRNA ion expression ions is measured by the invasive process of delivery, especially in vivo (13). Currently, methods are designed to deliver shRNA expression genes including cationic lipids and liposomes, proteins and the immune system. However, many websites limit the effectiveness of these methods to people. The use of antibodies has been shown to be an effective means of transmitting genes to many cells, though they do elicit specialized immune responses that can reduce p ‘clinical practice. Among non-invasive technologies, ultrasound-targeted microbubble depletion (UTMD) has emerged as a new, promising field for specialized field medicine and genetic in vitro and in vivo, providing in large quantities called sonoporation, to allow direct transmission to cells. (14–16). Special efforts have been made to attract siRNA expression utilized by UTMD to inhibit gene expression in vitro and in vivo (17 – 20). Wang et al (21) found that UTMD has the ability to transfer the remaining siRNA into SKOV-3 cells, which inhibit the expression of survival and induce apoptosis. This technology offers a promising new way of delivering siRNA in vitro. In our previous study (22), shRNA-survivin was incorporated into HeLa cells following UTMD, and we examined a reduction in survival expression. It is said that this process inhibits gene expression and prevents cell apoptosis. Furthermore, in a previous in vitro experimental study (23), we tried to solve a major problem caused by the use of a non-invasive genetic UTMD system (a combination of ultrasound signals) and liposome microbubbles) and PEI, especially transmission. of surviving shRNA. However, UTMD mechanism for shRNA in vitro delivery has not been improved, and mechanisms for apoptosis and effect using UTMD protocol and shRNA expression have not been studied.
In the present study, we investigated whether the different types of shRNA targeted survivors have the potential to be driven by the UTMD process. In fact, UTMD parlors of the shRNA in vitro delivery system are being upgraded. In addition, the effects of genetic inhibition and apoptosis activation, which we have not done before, were investigated. The results show that optimal radiation techniques allow for high transfer efficiency and do not affect the stability of plasmid DNA. UTMD coordinates the expression of a highly resilient mRNA protein, and induces cell apoptosis.
Human Hepatocellular Cells (HeLa) was obtained from the American Atomic Society (ATCC) and incorporated into Dulbecco’s modified Eagle’s medium (DMEM) supplement with 10% serum-free fetal bovine serum (Invitrogen Biotechnology, Shanghai , China). Cultures grow at 37 ° C in cold climates, with 5% CO2.
Examples of genetically modified oligonucleotides DNA (GenBank accession no. NM_001168) were developed and engineered in our previous study : survivin-shRNA1 (ori, 5′-GATCCG ACTGGAAGAAAGAGCCTCTAAGAGTTTCTCTT survivin-shRNA3 (ori, 5′-GATCCGGGACCACCGCATCTCTACATATAA GGATGTAGAGATGCGGTGGTCCTTTTTTTGGAAG-3 ′). These two fiber oligonucleotides are embedded in the P6IREN-DNR-DsRed-Express (BD Biosciences Clontech, USA) line of the P6IREN-DNR-DsRed-Express line in the BamHI and EcoRI area. Plasmids that can be reused for drying were extracted and purified using Qiaquick (Qiagen, Crawley, UK). Special shRNA trackers called pSIREN / S1, pSIREN / S2 and pSIREN / S3, respectively. Similarly, an unstable control vector is created (pSIREN / con). Temporary changes have been made using Lipofectamine ™ 2000 (Invitrogen Biotechnology, Shanghai, China), as described earlier (24, 25).
A) Cell Cycle Analysis By Flow Cytometry Based On Propidium Iodide…
In short, cells are harvested with 0.25% trypsin digestion after reaching 80-90% confluence and re-introduced into DMEM media (300 / l / well) in quantities of 1 × 107 cell / ml (26). SonoVue microbubbles (Bracco, Milan, Italy) are used to promote cavitation and then regenerate with saline solution within a mixture of 25-30 particles / cell, and are gradually added to stop the cell before p ultrasound imaging (25). UTMD tests performed on the signal tank and transducer (Accusonic, Metron Medical Australia Pty., Ltd.) were performed at the application center at the bottom of the tank, as described earlier (27).
Plasmid (last lethal dose 25 μg / ml) was added to the cell suspension prior to ultrasound imaging. Cells were exposed to several ultrasound power (0.4, 1.0, 1.6 and 2.2 W / cm2) at several times (1 and 3 min), at 10 or 20% of the operating time (DC) and frequency a 1 MHz, n. = 6 for each parameter.
Appearance of a modified red fluorescence protein (RFP) cell is observed with a different fluorescence microscope (Olympus IX71, Japan) after 48 (28) hours. Cells were then harvested, collected, and repositioned with phosphate-buffered salt (PBS). Approximately 1 × 105 cells were detected from each sample for transmission analysis, including 488-nm lavelength light and 585 ± 42 nm emission beam to detect red fluorescence using flow cytometry (FACSCalibur, Becton-Dickinson, USA). Transplantation was evaluated as the number of cells expressing RFP for the total number of cells involved.
DNA Plasmid (100) l) or SonoVue / DNA derivatives are included in 6 wells of 96 color plates, as described earlier (15), and are exposed to ultrasound (0.4, 1.0, 1.6 and 2.2 W / cm2; DC , 20). %; exposure time, 3 min) with or without the addition of SonoVue microbubbles. Gel electrophoresis test is performed immediately after ultrasound.
Cell Cycle Analysis
Cells were harvested 48 hours after transplantation, and all RNA was secreted by the TRIzol (Invitrogen) reagent according to the manufacturer’s instructions. RNA is converted to cDNA with a flexible divorce application (Promega, Madison, WI, USA). Special primers are developed based on human cDNA protocols using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and developed by Invitrogen. Sequence of primers are: first, 5′-ACCACCGCATCTCTACACATTC-3 ‘; back, 5 “-AGCTG GCTCGTTCTCAGTG-3”; and the forecasting market is 113 bp. GAPDH is used as an internal stimulant and its mRNA is increased by primers: first, 5′-GAAGGTGAA GGTCGGAGTC-3 ′; rear, 5’-GAAGATGGTGATGGG ATTTC-3 ‘; and the forecasting market is 226 bp. PCR performance enhancement was performed and the resulting products were illuminated by 1% agarose gel containing ethidium bromide. Survival and GAPDH potency were tested using Quantity One software (Bio-Rad, Hercules, CA, USA). Conditions related to the term “survivin” are presented as part of survivin / GAPDH.
Cells were washed twice with cold PBS before being inserted into the cell lysis pool. Cells were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and deposited in nitrocellulose membranes (Millipore, Billerica, MA, USA). The Immunoblot test was performed according to method (24). Disorders are developed using chemiluminescence enhancement. The survivin related conditions are presented as the survivin / act-actin ratio.
Cells were tested for flow cytometry (FACSCalibur, BectonDickinson) followed by double contamination of cells containing annexin V and 7-AAD (Jinmei Biotech Co. Ltd., Wuhan, China). Early apoptotic cells are contaminated with only FITC-labeled annexin-V, while necrotic and lateral cells are contaminated with annexin.
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