| Background and objectiveIn recent years, the morbidity of lung cancer which compared with other cancers has been increasing with a rapid rate in our country.At present, lacking the specific and sensitive early diagnosis and therapeutic molecules targets of lung cancer. Cancer stem cells(CSC) are concidered to be the sources of tumor formation,development,recurrence and metastasis, searching lung cancer stem cells might become to be one new potential breakthrough point for early diagnosis and therapeutic of lung cancer. Substantial studies indicate that micro RNA(mi RNA) is widely involved in the tumor occurrence, development, angiogenesis, metastasis, recurrence and drug resistance, etc. Identifying the related micro RNAs of lung cancer cells or lung cancer stem cells might become to be an important strategy for early diagnosis and therapeutic of lung cancer. Molecular beacon is one kind of oligonucleotide sequences labeled with fluorescence probe which forms the hairpin structure. It is a very sensitive tools for DNA, RNA and micro RNA detection and has been widely used in gene quantitative analysis, disease diagnosis and living imaging fields.Chitosan is a kind of natural cationic polysaccharide which has been widely used for delivery plasmid DNA and small interference RNA(si RNA) and other genes. Chitosan nanoparticles certify the gene transfection in vivo and vitro with higher safety and effectiveness. Due to the difference of enhanced permeability and retention effect(EPR) in different tissues, it is not an ideal strategy to utilize the passive targeting characters of nano-materials. Tumor cells surface have a lot of specific receptors or antigens that can be used to distinguish normal cells. We could modify specific peptide and monoclonal antibodies at the nano-materials surface through the specific binding to the receptors or antibodies, we can realize the tumor targeting diagnosis and treatments, such as the somatostatin receptor 2(SSTR2). In our researches, lung cancer stem cells(LCSC) were sorted and identified form the non-small cell lung cancer(NSCLC) model. According to the OCT could specific binding to the SSTR2, by the molecular beacon, nanotechnology, and modified targeted peptides(Octreotide, OCT) at the nano-matreials surface, our aim was using the CS-MB-OCT nanoparticles to detect and image the micro RNAs that high expressed in lung cancer cells or lung cancer stem cells, and therefor imaging the cancer cells. It will be a new idea and technology for early diagnosis of lung cancer.Materials and methods1. Sorted the CD133 + CD326 + double positive cells from A549 cells by flow cytometry, and identified their stem character. The mi R-155 expression level was detected by real time PCR. 2. Designing and synthesizing the mi R-155 molecular beacon(mi R-155 MB) which was modified by lock nucleic acid according to the mi R-155 sequence. Designing the random sequence molecular beacon(RS MB) which was not complementary with any other sequences as the negative control. The specificity and sensitivity of the designed molecular beacon was detected by the hybridization assay. 3. Synthesizing the chitosan and molecular beacon(CS-MB) complexes self-assembly and testing the complexes particle size, zeta potential, dispersion and anti-DNase I properties.Chitosan nanoparticles were adopted to deliver the mi R-155 MB and the RS MB was made as the negative control. The laser confocal microscopy was used to detect and image the mi R-155 in NSCLC and LCSC. The mi R-155 expression level was verified by q RT-PCR. PC-3 cells were used as the positive control cells. Furthermore, detecting and imaging the mi R-155 in NSCLC and LCSC were also researched in vivo. The fluorescence intensity and transfection efficiency were also compared between the Chitosan nanoparticles carrier and commercial si PORT liposomes carrier. 4. The SSTR2 expression in NSCLC cell and tumor tissues was detected by immunofluorescence. Synthesized the chitosan and molecular beacon complexes modified with octreotide(CS-MB-OCT) by chemical modified methods and detected its physicochemical characteristics. Detecting and imaging the mi R-155 were also studied by CS-MB-OCT in vitro and in vivo. The fluorescence intensity and transfection efficiency were also compared among the si PORT transfection agent, CS-MB and CS-MB-OCT.Results1. The CD133 + CD326 + double positive cells formed the sphere in stem cell culture medium, 1×104 cells could form the subcutaneous transplanted tumors. The stem genes(CD133,CD326,OCT-4,Nanog) expression levels were 3 times higher than A549 cells, and the mi R-155 expression was 2.63 times higher than A549 cells. 2. Hybridization assay showed that the fluorescence intensity in the mi R-155+mi R-155 MB group was 55 times higher than that in the only mi R-155 MB group, and the fluorescence intensity in the RS+RS MB group was 41 times higher than that in the only RS MB group, which showed the MB targeted reciprocal sequences effectiveness and sensitiveness. 3. Synthesizing the CS-MB at the weight rate as 7:1, the sizes of the CS-MB nanoparticles were 15-80 nm, with an average of 31.95±3.28 nm. The mean zeta potentials of CS-MB was+20.1±2.14 m V. The CS-MB can be resistant to the degradation of DNase I and suitable for gene delivery. At the cellular level, the red fluorescence could be detected in the NSCLC and LCSC cytoplasm after CS nanoparticles transfect the mi R155 MB, and the fluorescence intensity was consistent with the level of mi R-155 expression determined by q RT-PCR. What is more, the red fluorescence was no detected in the RS MB group. At the live animals’ level, the red fluorescence could be detected in the subcutaneous transplanted tumors and no red fluorescence was detected in the RS MB group. The CS nanoparticles transfection efficiency was about 75%–85%, and showed higher fluorescence intensity and transfection efficiency, compared to the commercial si PORT liposome reagents. 4. The SSTR2 expressed in NSCLC cell and tumor tissues and could be targeted by octreotide. Synthesizing the chitosan and molecular beacon complexes modified with octreotide(CS-MB-OCT) by chemical modified methods. The sizes of the CS-MB-OCT nanoparticles were 30-130 nm, with an average of 82.28±1.24 nm. At the live animals’ level, the CS-mi R155 MB-OCT could detect and image the mi R-155 in the subcutaneous and lung transplantation tumor of NSCLC. Compared to the CS-mi R155 MB, the CS-mi R155 MB-OCT showed higher fluorescent intensity and transfection efficiency.Conclusion1. The CD133+CD326+ double positive cells had the stem cell properties and expressed higher mi R-155 than A549 cells. The mi R- 155 MB modified by lock nucleic acid could identify mi R- 155 effectively and sensitively. 2. The CS-mi R155 MB complexes can detect and image the mi R-155 at the cellular level and animal level. By identifying the mi R-155 we could image the NSCLC or LCSC cells in vitro and in vivo. 3. Compared to the CS-mi R155 MB complexes, CS-mi R155 MB-OCT could specific bind the somatostatin receptors, targeted the cancer cells and better detected and imaged the mi R-155 at the cellular level and animal level. Our researches will be a new idea and technology for early diagnosis of lung cancer. |
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