Percutaneous dynamic release in stress position by acupotomy in treating severe scapulohumeral periarthritis / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the clinical efficacy of acupotomy stress position percutaneous dynamic release for severe shoulder periarthritis.</p><p><b>METHODS</b>From April 2012 to August 2016, 160 patients with severe shoulder periarthritis were randomly divided into treatment group and control group. Among them, 80 patients in treatment group were treated with acupotomy stress position percutaneous dynamic release including 32 males and 48 females with an average of(52.47±9.04)years old ranging from 40 to 74 years old;the courses of disease was(20.72±9.55)months on average. The other 80 patients in control group were treated with simple joint loosening according to Maitland technique in grade III-IV therapy, once a day, 15 to 20 min each time, and 10 d for 1 course, for a total of 2 courses, including 33 males and 47 females with an average of (53.19±10.18) years old ranging from 42 to 75 years old; the average course of disease was (21.98 ±8.99) months. After operation, the shoulder muscles training and shoulder joint activity training were routinely conducted, the treatment lasted for 3 weeks. The visual analogue scale(VAS) and Constant-Murley shoulder function score were observed and compared between the two groups before treatment and 3 weeks, 3, 6 months after treatment.</p><p><b>RESULTS</b>The VAS scores of the treatment group at 3 weeks, 3 and 6 months after treatment were all lower than those of the control group(<0.05). The shoulder joint function Constant-Murley scores of the treatment group at 3 weeks, 3 and 6 months after treatment were higher than those of the control group (<0.05); the result was excellent in 59 cases, good in 18 cases, fair in 3 cases in the treatment group; excellent in 15 cases, good in 31 cases, fair in 23 cases, poor in 11 cases in the control group, and the difference between the two groups was statistically significant(<0.01).</p><p><b>CONCLUSIONS</b>Treatment of severe shoulder periarthritis with acupotomy stress position percutaneous dynamic release can obviously improve the shoulder joint function and pain, according to the different parts of the shoulder joint pain and function limitation, the corresponding shoulder stress and body position should be designed and maintained during the treatment process, and the angle of stress position gradually increased by loosening the adhesion, which is the key to ensure the curative effect.</p>

MiR-135b promotes proliferation of endometrial carcinoma cells by targeting FOXO1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of miR-135b in endometrial carcinoma and the mechanism by which miR-135b promotes the proliferation of endometrial cancer cells.</p><p><b>METHODS</b>The expressions of miR-135b and FOXO1 were using RT-PCR detected in 22 fresh endometrial cancer tissues and paired adjacent tissues and also in endometrial cancer cell lines JEC, Ishikawa, HEC-1-B, and RL-952. The RL-952 and Ishikawa cell lines were transfected with miR-135b mimics or inhibitors, and the changes in their proliferative activity were detected with MTT assay; the expressions of FOXO1 mRNA and protein were detected by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>The expression of miRNA135b was significantly up-regulated and FOXO1 expression was down-regulated in endometrial carcinoma tissues as compared with the adjacent tissues (P<0.05). The mRNA expression of miR-135b was negatively correlated with the expression of FOXO1 in endometrial carcinoma. In RL-952 and Ishikawa cell lines, transfection with miR-135b mimics obviously promoted the cell proliferation (P<0.05). Up-regulation of miR-135b significantly decreased the expressions of FOXO1 protein and mRNA (P<0.05), and down- regulation of miR-135b increased FOXO1 expressions (P<0.05).</p><p><b>CONCLUSIONS</b>MiR-135b plays an important role in the occurrence and development of endometrial carcinoma partially by regulating its target gene FOXO1.</p>

Effect of miR-101 on biological characteristics of colorectal cancer cell line SW620 / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of miR-101 on biological behaviors of colorectal cancer cell line SW620.</p><p><b>METHODS</b>CCK-8 method, colony formation assay, cell cycle analysis and apoptosis analysis were applied to assess the effects of miR-101 on cell proliferation, invasion and apoptosis of SW620 cells.</p><p><b>RESULTS</b>Over-expression of miR-101 in SW620 cells significantly suppressed the cell proliferation and attenuated the colony-forming ability of the cells. Flow cytometry showed that over-expression of miR-101 in SW620 cells caused obvious cell cycle arrest in G2/M and 1/ phases, and significantly increased the cell apoptosis rate.</p><p><b>CONCLUSION</b>Over-expression of miR-101 can inhibit the proliferation, cause cell cycle arrest and promote apoptosis of colorectal cancer SW620 cells.</p>

Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335.</p><p><b>METHODS</b>The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the expression of RASA1 protein in SW620 cell lines.</p><p><b>RESULTS</b>The recombinant lentiviral vector PLVTHM-miR335, psiCHECK-2-RASA1 and the mutation expression vector psiCHECK-2-RASA1-Mut were successfully constructed. Dual-luciferase reporter assay showed that miR-335 decreased luciferase activity in cells co-transfected with psiCHECK-2-RASA1. The expression of miR-335 in SW620 cells infected with the lentivirus PLVTHM-miR335 was significantly increased, but the expression of RASA1 showed only slight changes. Overexpression of miR-335 suppressed the expression of RASA1 protein in SW620 cells.</p><p><b>CONCLUSION</b>We have successfully constructed the lentiviral vector containing mir-335 gene and a SW620 cell line with miR-335 overexpression. MiR-335 can suppress RASA1 gene expression by targeting the specific sequence of RASA1 3'UTR.</p>

Long-term culture and identification of CD133⁺ hematopoietic progenitor cells from human umbilical cord blood / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate CD133(+) hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics.</p><p><b>METHODS</b>CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system, and the cells were detected by flow cytometry. Four methods were used for culturing cells. After 8 weeks' culture, cytomorphology, flow cytometry, immunocytochemistry and immunofluorescence assay were used to identify the characteristics of the stem cells.</p><p><b>RESULTS</b>Over 80% of CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system. The cells were effectively expanded using optimized serum-free medium after 8 weeks of cell culture, whereas the cells in other media differentiated into adherent cells in a poor state.</p><p><b>CONCLUSION</b>The optimized serum-free medium allows effective expansion of CD133(+) hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.</p>

Touchdown PCR and overlap extension PCR for generating CD133(+) cancer stem cell-selective adenovirus vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133(+) SW480 cells in vitro.</p><p><b>METHODS</b>The two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR. The product obtained was identified by sequencing and inserted into prokaryotic expression vector pEGFP-N1. The product, pEGFP-N1 KNOBδHI, contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop. The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR, then identified by sequencing and inserted into pNEB193, resulting in pNEB-F5. CD133(+) SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope.</p><p><b>RESULTS</b>The target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR, but not by common PCR. Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133(+) SW480 cells.</p><p><b>CONCLUSION</b>Compared with common PCR, touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133(+) cancer stem cell-selective adenovirus type 5 vector, which provides carriers for tumor-targeted gene therapy.</p>

Selection and identification of specific-binding peptides for cancer stem cell surface marker CD133 / 南方医科大学学报

<p><b>OBJECTIVE</b>To select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library.</p><p><b>METHODS</b>With a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.</p><p><b>RESULTS</b>After 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133.</p><p><b>CONCLUSION</b>We have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.</p>

Role of vascular endothelial growth factor receptor 1-positive hematopoietic progenitor cell clusters in human colorectal carcinoma metastasis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the relation of vascular endothelial growth factor receptor 1 (VEGFR1)-positive hematopoietic progenitor cell with the metastasis of human colorectal carcinoma.</p><p><b>METHODS</b>Human colorectal cancer cells SW480/M5 were implanted into BALB/c nude mice, and the tumor tissue blocks were re-implanted into the colon of nude mice. The quantity and percentage of VEGFR1-positive hematopoietic progenitor cells and human colorectal cancer cells in the pre-metastatic location were observed with flow cytometry, and the expression of metastasis-related factors was detected using Western blotting.</p><p><b>RESULTS</b>Bone marrow-derived hematopoietic progenitor cells expressing VEGFR1 were found in the common pre-metastatic sites and formed cell clusters before the arrival of the tumor cells, but these cells were not detected in nude mice without tumor implantation. In the course of tumor metastasis, the expression of the proteinases including matrix metalloproteinase 9 and stromal cell-derived factor 1 gradually intensified within the metastatic niche.</p><p><b>CONCLUSION</b>The formation of VEGFR1-positive hematopoietic progenitor cellular clusters is accompanied by the metastasis of human colorectal cancer, and may enhance the expression of metastasis-related factors, suggesting its important role in the invasion and metastasis of colorectal cancer.</p>

Establishment of a whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice through serial passage in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice and observe the development and metastasis of breast cancer by real-time imaging.</p><p><b>METHODS</b>pEGFP-N1 plasmid was transfected into human breast cancer cell line MDA-MB-231 to obtain pEGFP-MDA-MB-231 cells that emitted fluorescence. pEGFP-MDA-MB-231 cells were inoculated orthotopically in BALB/C nude mice and cultured in vivo through serial passage, thereby establishing the mouse model bearing tumors with high hepatic metastasis potential. The fluorescence emitted from the tumors was quantitatively detected and imaged with a fluorescence stereo microscope for real-time visualization of the tumor growth and metastasis.</p><p><b>RESULTS</b>The transfected breast cancer cells stably and efficiently expressed EGFP. After inoculation of the transfected cells in nude mice, 20% of the first-generation cells showed hepatic metastasis, and the rate increased to 80% among the second-generation and up to 100% among the third-generation cells. The reliability of this visualization model was validated with conventional pathological methods.</p><p><b>CONCLUSION</b>The whole-body visualization model bearing breast cancer with high hepatic metastasis potential provides a reliable means for studying the mechanisms of hepatic tumor metastasis, and can be instrumental in the exploration of novel means for breast cancer treatment.</p>