[Establishment of a mouse model of testicular Occludin gene knockout based on the CRE/LOXP system].

OBJECTIVE: To establish a testicular Occludin gene knockout model in mice and observe the phenotypic changes. METHODS: Occludin-floxed (genotype Floxp/-) mice were constructed based on the Cre/loxp system, which were cross-bred with AQP2-cre (genotype Cre/-) mice to derive Occludin knockout mice (Genotype Floxp/Floxp Cre/-). The genotype of the F1 knockout mice was identified by PCR and Southern blot technology. The expression of the Occludin protein in the knockout mice was determined by qPCR, Western blot and immunohistochemistry to verify the success of the modeling. Comparisons were made in the sperm count between the model and normal mice, followed by analysis of their fertility. RESULTS: The target mice of Occludin knockout were successfully constructed, which, compared with the normal controls, showed significantly down-regulated expression of the occludin protein, decreased sperm count and reduced fertility (P < 0.05). CONCLUSIONS: Occludin gene knockout mice were successfully constructed, and deletion of Occludin affects the reproductive function of the mice.

[SP1 identified as a transcription factor of miR-122-5p].

OBJECTIVE: To study the transcription factors of the spermatogenesis-related promoter mir-122-5p. METHODS: SP1 and GATA4 were predicted as the possible transcription factors of the mir-122-5p promoter by bioinformatics analysis, followed by construction of the double luciferase pGL3-mir-122-5p promoter vector, pcDNA3.1 (+) -SP1 expression vector and pcDNA3.1 (+) -GATA4 expression vector, respectively. The pcDNA-SP1+pGL3-basic mixture plasmid and pcDNA-SP1+ pGL3-miR-122-5p promoter mixture plasmid, pcDNA-GATA4+pGL3-basic mixture plasmid and pcDNA-GATA4+pGL3-miR-122-5p promoter mixture plasmid were transferred into 293T cells. The enzyme activity was detected the Dual-Luciferase Reporter Assay System. RESULTS: The fluorescence value of the pcDNA3.1+pGL3-miR-122 promoter was 0.0362 ± 0.0004, significantly higher than that of the pcDNA3.1+pGL3-basic group (P < 0.05), indicating the successful construction of the mouse miR-122-5p promoter luciferase reporter plasmid. The fluorescence value was markedly higher in the pcDNA -SP1 + pGL3-miR-122-5p promoter than in the pcDNA -SP1+pGL3-basic group, suggesting that the transcription factor SP1 could promote the transcription of miR-122. There was no statistically significant difference in the fluorescence value between the pcDNA -gata4+pGL3-basic transfection and pcDNA -GATA4+pGL3-miR-122-5p promoter transfection groups, indicative of the inability of GATA4 to promote the transcription of miR-122-5p. CONCLUSIONS: The transcription factor SP1, rather than GATA4, can promote the transcription of miR-122-5p.

[Construction of RNA interfering lentiviral vector of occludin and its role in tight junctions].

OBJECTIVE: To investigate the role of occludin in tight junction (TJ) in vitro. METHODS: We constructed RNA interfering lentiviral vectors and transfected them into TM4 cells. Then we detected their inhibitory effect on occuldin by RT-PCR and Western blot and analyzed the role of occuldin in TJ using an in vitro TJ cell model. RESULTS: The pLenti 6.3-EGFP-occludin-miR expression vector was successfully constructed. The results of RT-PCR and Western blot showed that pLenti 6.3-EGFP-occludin-miR-3 significantly inhibited the expression of occludin (P < 0.05), which was remarkably lower than in the blank control and the pLenti 6.3- EGFP transfection group (0.7534 ± 0.089 vs 1.000 and 1.056 ± 0.025, P < 0.05). The expression of occludin was markedly suppressed and the tightness of tight junctions decreased in the TM4 cells transfected with pLenti 6.3-EGFP-occludin-miR-3. CONCLUSIONS: The pLenti 6.3-EGFP-occludin-miR expression vector was successfully constructed, and occludin is one of the functional proteins that maintain tight junctions.

Therapeutic effects of the TST36 stapler on rectocele combined with internal rectal prolapse.

BACKGROUND: The most common causes of outlet obstructive constipation (OOC) are rectocele and internal rectal prolapse. The surgical methods for OOC are diverse and difficult, and the postoperative complications and recurrence rate are high, which results in both physical and mental pain in patients. With the continuous deepening of the surgeon's concept of minimally invasive surgery and continuous in-depth research on the mechanism of OOC, the treatment concepts and surgical methods are continuously improved. AIM: To determine the efficacy of the TST36 stapler in the treatment of rectocele combined with internal rectal prolapse. METHODS: From January 2017 to July 2019, 49 female patients with rectocele and internal rectal prolapse who met the inclusion criteria were selected for treatment using the TST36 stapler. RESULTS: Forty-five patients were cured, 4 patients improved, and the cure rate was 92%. The postoperative obstructed defecation syndrome score, the defecation frequency score, time/straining intensity, and sensation of incomplete evacuation were significantly decreased compared with these parameters before treatment, and the differences were statistically significant (P < 0.05). The postoperative anal canal resting pressure and maximum squeeze pressure in patients decreased compared with before treatment, and the differences were statistically significant (P < 0.05). The initial and maximum defecation thresholds after surgery were significantly lower than those before treatment, and the differences were statistically significant (P < 0.05). The postoperative ratings of rectocele, resting phase, and defecation phase in these patients were significantly decreased compared with those before treatment, and the differences were statistically significant (P < 0.05). CONCLUSION: The TST36 stapler is safe and effective in treating rectocele combined with internal rectal prolapse and is worth promoting in clinical work.

Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis.

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.

Consensus statement on diagnosis and treatment of cervical ossification of posterior longitudinal ligament from Asia Pacific Spine Society (APSS) 2020.

PURPOSE: The study aimed to develop an evidence-based expert consensus statement on diagnosis and treatment of cervical ossification posterior longitudinal ligament (OPLL). METHOD: Delphi method was used to perform such survey, and the panel members from Asia Pacific Spine Society (APSS) 2020 were invited to answer the open-ended questions in rounds 1 and 2. Then the results were summarized and developed into a Likert-style questionnaire for voting in round 3, and the level of agreement was defined as 80%. In the whole process, we conducted a systematic literature search on evidence for each statement. RESULTS: Cervical OPLL can cause various degrees of neurological symptoms, an it's thought to be more common in Asia population. CT reconstruction is an important imaging examination to assist diagnosis and guide surgical choice. Segmental, continuous, mixed, and focal type is the most widely used classification system. The non-surgical treatment is recommended for patients with no or mild clinical symptoms, or irreversible neurological damage, or failed surgical decompression, or condition cannot tolerant surgery, or refusing surgery. As OPLL may continue to develop gradually, surgical treatment would be considered in their course inevitably. The surgical choice should depend on various conditions, such as involved levels, thickness, and type of OPLL, skill-experiences of surgeons, which are listed and discussed in the article. CONCLUSION: In this statement, we describe the clinical features, classifications, and diagnostic criteria of cervical OPLL, and review various surgical methods (such as their indications, complications), and provide a guideline on their choice strategy.

[Deletion of extracellular loops of occludin affects the tight junction of TM4 cells in mice].

OBJECTIVE: To analyze the functions of the two extracellular loops of occludin in the tight junction of TM4 cells in mice. METHODS: Using genetic engineering, we separately or simultaneously deleted two extracellular loops of occludin, cloned the three occludin genes without extracellular loops into the pcDNA3.1 expression vector, and transfected them into TM4 cells. Then we determined the expression of occludin by RT-PCR and Western blot, and analyze the effects of the extracellular loops of occludin on the tight junction of the TM4 cells with the in vitro cell line model. RESULTS: The results of sequencing showed that the expression vector of pcDNA3.1 - occludin Δ OCC1, pcDNA3.1 - occludin Δ OCC2 and pcDNA3.1 - occludin Δ OCC1 + OCC2 was constructed successfully. The mRNA and protein expressions of occludin in the non-extracellular loop groups were significantly higher than in the control group. Both the extracellular loops of occludin increased the tight junction of the TM4 cells. The macromolecular permeability in the TM4 cells was significantly lower in the pcDNA3.1 - occludin Δ OCC1 than in the pcDNA3.1 - occludin Δ OCC2 group (P < 0.05), indicating a higher impact of the second than the first extracellular loop on the tight junction of the TM4 cells. CONCLUSIONS: Both of the two extracellular loops of occludin can affect the tight junction of TM4 cells, the second even more significantly than the first one.

Effects of Length and Loop Composition on Structural Diversity and Similarity of (G3TG3NmG3TG3) G-Quadruplexes.

A G-rich sequence containing three loops to connect four G-tracts with each ≥2 guanines can possibly form G-quadruplex structures. Given that all G-quadruplex structures comprise the stacking of G-quartets, the loop sequence plays a major role on their folding topology and thermal stability. Here circular dichroism, NMR, and PAGE are used to study the effect of loop length and base composition in the middle loop, and a single base difference in loop 1 and 3 on G-quadruplex formation of (G3HG3NmG3HG3) sequences with and without flanking nucleotides, where H is T, A, or C and N is T, A, C, or G. In addition, melting curve for G-quadruplex unfolding was used to provide relatively thermal stability of G-quadruplex structure after the addition of K+ overnight. We further studied the effects of K+ concentration on their stability and found structural changes in several sequences. Such (G3HG3NmG3HG3) configuration can be found in a number of native DNA sequences. The study of structural diversity and similarity from these sequences may allow us to establish the correlation between model sequences and native sequences. Moreover, several sequences upon interaction with a G-quadruplex ligand, BMVC, show similar spectral change, implying that structural similarity is crucial for drug development.

Temporal and spatial surveys of polybromodiphenyl ethers (PBDEs) contamination of soil near a factory using PBDEs in northern Taiwan.

Polybrominated diphenyl ethers (PBDEs), previously commonly used as flame retardants, should be monitored in the environment since some are listed as persistent organic pollutants. A contaminated site near a northern Taiwan factory using decabrominated diphenyl ether (deca-BDE) was identified based on a vegetable PBDEs monitoring project in 2013. The subsequent spatial and temporal survey of that contaminated site shows the contamination ingredients in soils were close to ones used by the factory, indicating that contamination was from the factory, possibly through an exhaust vent. The average concentration of deca-BDE in the main contaminated soil was 615 µg/kg d. w. (dry weight) soil in 2015, slightly decreasing to 604 µg/kg d. w. soil in 2016, increasing to 844 µg/kg d. w. soil in 2017, and then slightly decreasing to 670 µg/kg d. w. soil in 2018. The slight change of deca-BDE and the minor change in low brominated congener level indicate a low degradation rate. The contamination of peripheral sites was around 5000 µg/kg d. w. soil for one PBDEs sampling site that was higher than those around or within the main contaminated farm, indicating serious pollution. Concentrations of PBDEs in different soil depths show that depth 2-15 cm accounted for the greatest PBDEs accumulation, indicating that deca-BDE pollution had been present over time and transported into deeper soil. There can be PBDEs uptake by crops consumed by humans, as shown in our previous studies, so continuous monitoring of PBDEs in this site is important and treatments should be established urgently.

16 Gb/s PAM4 UWOC system based on 488-nm LD with light injection and optoelectronic feedback techniques.

A 16 Gb/s four-level pulse amplitude modulation (PAM4) underwater wireless optical communication (UWOC) system based on 488-nm laser diode (LD) with light injection and optoelectronic feedback techniques is proposed and successfully demonstrated. Experimental results show that such a 1.8-GHz 488-nm blue light LD with light injection and optoelectronic feedback techniques is enough forceful for a 16 Gb/s PAM4 signal underwater link. To the authors' knowledge, this study is the first to successfully adopt a 488-nm LD transmitter with light injection and optoelectronic feedback techniques in a PAM4 UWOC system. By adopting a 488-nm LD transmitter with light injection and optoelectronic feedback techniques, good bit error rate performance (offline processed by Matlab) and clear eye diagrams (measured in real-time) are achieved over a 10-m underwater link. The proposed system has the potential to play a vital role in the future UWOC infrastructure by effectively providing high transmission rate (16 Gb/s) and long underwater transmission distance (10 m).

p53 suppresses 14-3-3γ by stimulating proteasome-mediated 14-3-3γ protein degradation.

14-3-3 proteins are a family of highly conserved polypeptides that interact with a large number of proteins and play a role in a wide variety of cellular processes. 14-3-3 proteins have been demonstrated overexpressed in several cancers and serving as potential oncogenes. In a previous study we showed one isoform of the 14-3-3 family, 14-3-3γ was negatively regulated by p53 through binding to its promoter and inhibiting its transcription. In the present study we investigated both p53 and 14-3-3γ protein levels in human lung cancerous tissues and normal lung tissues. We found 14-3-3γ expression correlated to p53 overexpression in lung cancer tissues. Ecotopic expression of wild-type p53, but not mutant p53 (R175H) suppressed both endogenous and exogenous 14-3-3γ in colon and lung cancer cell lines. Further examination demonstrated that p53 interacted with C-terminal domain of 14-3-3γ and induced 14-3-3γ ubiquitination. MG132, a specific inhibitor of the 26S proteasome, could block the effect of p53 on 14-3-3γ protein levels, suggesting that p53 suppressed 14-3-3γ by stimulating the process of proteasome-mediated degradation of 14-3-3γ. These results indicate that the inhibitory effect of p53 on 14-3-3γ is mediated also by a post-transcriptional mechanism. Loss of p53 function may result in upregulation of 14-3-3γ in lung cancers.

Biotransformation of trinitrotoluene (TNT) by Pseudomonas spp. isolated from a TNT-contaminated environment.

The compound 2,4,6-trinitrotoluene (TNT) is a secondary explosive widely used worldwide for both military and civil purposes. As a result, residual TNT has been detected as an environmental pollutant in both soil and groundwater. The authors have isolated several microbial strains from soil contaminated with TNT by enrichment culture techniques using TNT as a carbon, nitrogen, and energy source. The contaminated soil contained approximately 1860 ppm TNT measured by high-performance liquid chromatography (HPLC). The initial identification of these isolates was determined by 16S rRNA gene comparison. The isolates mainly included species belonging to the genus Pseudomonas. Two strains (Pseudomonas putida strain TP1 and Pseudomonas aeruginosa strain TP6) were selected for further examination. Both strains demonstrated the ability to grow on the medium containing TNT as a carbon, energy, and nitrogen source and also clearly demonstrated the ability to degrade TNT. More than 90% of the TNT in the growth medium was degraded by both strains after 22 d incubation, as determined by HPLC. Additionally, the resting cells of P. putida TP1 and P. aeruginosa TP6 both significantly displayed the ability to transform (metabolize) TNT.

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer.

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloid-derived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/mL, P = 0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P = 0.0162). The frequencies of Th17 cells (CD4⁺IL-17A⁺) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P = 0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P = 0.0041 and P = 0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P = 0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r = -0.1725, P = 0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r = 0.6404, P = 0.0031) and TNF-α (r = 0.7646, P = 0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r = 0.6782, P = 0.0007) and TNF-α (r = 0.7633, P < 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

[Surgical options and clinical outcomes of cervical ossification of the posterior longitudinal ligament].

OBJECTIVE: To discuss the options and clinical outcomes of surgical treatment of cervical ossification of the posterior longitudinal ligament (OPLL). METHODS: From January 2006 to December 2010, 63 patients of cervical OPLL treated surgically by same team were retrospectively analyzed. There were 49 males, 14 females, with a mean age of 56.2 years (from 38 to 76 years). The duration of symptoms ranged from 2 months to 20 years (mean 3.2 years). The ossified ligament was classified via sagittal and coronal images on CT scan. The cervical curvature was measured. Choice of surgical methods was determined according to the type of ossified ligament and cervical curvature. All complications occurred was recorded. The Japanese Orthopedic Association (JOA) scoring system was used to evaluate the neurological status. The severity of cervical pain was assessed with visual analogue scale (VAS) method. The results of preoperative, postoperative and follow-up were statistically compared with student t test. RESULTS: The JOA scores of 35 patients performed with anterior approach improved from 9.8 ± 2.3 preoperatively to 11.7 ± 2.1 at final follow-up (t = 3.28, P < 0.05). The VAS sore was significantly decreased at final follow-up compared with preoperative (t = 3.15, P < 0.05). While the JOA scores improved from 10.4 ± 2.7 preoperatively to 12.5 ± 2.3 at final follow-up (t = 3.81, P < 0.05) in 12 patients with laminectomy and from 9.7 ± 2.5 to 11.6 ± 2.6 in 14 patients with laminoplasty (t = 3.56, P < 0.05). The VAS score either in laminectomy or in laminoplasty has not significantly difference between preoperative, 3 month postoperative and final follow-up (P > 0.05). Two patients underwent a combined anterior-posterior approach procedure. Complications in patients included 1 patient of postoperative neurologic deterioration and 2 cases of cerebrospinal fluid leakage in patients with anterior approach, and 2 cases of C(5) nerve palsy in patients with laminectomy. CONCLUSIONS: Surgical options of cervical OPLL should be determined by detailed study of type and range of ossified ligament, as well as the cervical curvature of patients. Good neurological function can be expected in case of appropriate choosing the method for treating the cervical OPLL.

The ATP2A2 gene in patients with Darier's disease: one novel splicing mutation.

BACKGROUND: Darier's disease (DD) is a rare, inherited skin disorder characterized by warty papules and plaques over the seborrheic area, such as central trunk, flexures, scalp, and forehead. Mutations in ATP2A2 gene encoding the enzyme sarco/endoplasmic reticulum Ca(2+) ATPase type 2 are responsible for the disease. Here we report two Chinese families affected by DD with two ATP2A2 mutations. MATERIALS AND METHODS: DNA was extracted from the peripheral blood samples and then subjected to polymerase chain reaction amplification and direct automated DNA sequencing. RESULTS: A heterozygous G to T transition in the first nucleotide of intron 7 (c.630 + 1G>T) and G to A transversion at nucleotide 2898 in exon 20 of the ATP2A2 gene were identified in two pedigrees, respectively. The former mutation in the splice site is a novel mutation and is thought to lead to aberrant splicing and premature protein truncation. The latter has already been described, which leads to premature termination codons (PTC) (W966X). CONCLUSION: The results will contribute to the expanding database of ATP2A2 mutations in patients with DD and be useful for inherited counseling and prenatal examination for affected families.

Single-stage combined decompression for patients with tandem ossification in the cervical and thoracic spine.

STUDY DESIGN: Retrospective study of clinical outcomes of single-staged combined cervical and thoracic decompression for patients with tandem ossification (TO). OBJECTIVE: To describe primary clinical outcomes of this procedure. TO is introduced to described a double ossification lesion of the posterior longitudinal ligament (OPLL) or the ligament flavum (OLF) at the cervical, thoracic and lumbar spine. In clinical practice, cervical OPLL combined with thoracic OPLL or/and OLF are the most common types of TO. However, little is known about the clinical outcomes of surgical treatment and there is no consensus on the optimal treatment to this combined disorder. METHODS: Between January 2005 and December 2008, 15 patients of this complicated phenomenon were treated by single-staged combined cervical and thoracic decompression in conditions where patients' general condition allowed and individuals agreed on. Surgical intervention, perioperative complications, and clinical outcomes were reviewed in these 15 TO patients who were followed up for more than 2 years (range 2-5 years). Clinical symptoms were evaluated using the JOA scoring system and activity of daily life was evaluated by Nurick classification before surgery, at 6 months postoperatively, and at final follow-up. Patient satisfaction was determined at final follow-up. RESULTS: The mean blood loss was 1,553.3 ± 735.7 ml (range 700-2,900 ml) and the mean operation time was 280.7 ± 53.6 min (range 220-370 min). The important intraoperative and postoperative complications recorded in medical documents included CSF leakage, hematoma, C5 palsy and neurological deterioration. The JOA score was significantly higher 6 months after surgery (8.1 ± 1.8 points vs. 11.0 ± 1.6 points, p < 0.0001), and there was no significant change between 6 months after surgery and final follow-up (11.0 ± 1.6 points vs. 11.3 ± 2.1, p = 0.5894). The mean Nurick classification significantly improved from grade 3.6 ± 0.7 before surgery to grade 2.5 ± 0.9 at 6 months after surgery (p < 0.001), and well maintained as grade 2.3 ± 1.0 at final follow-up (p = 0.3343). Three patients had satisfaction scores of 3 points, 5 had scores of 2 or 1 point, and 2 had score of 0 point. Pearson correlation analysis showed a significant positive correlation between satisfaction score and JOA score (r = 0.6493, p = 0.0093), and a significant negative correlation between satisfaction score and Nurick classification (r = -0.5941, p = 0.0195). Besides, perioperative complications and progression of tandem ossification which needed revision surgery had significant adverse effect on patients' satisfaction. CONCLUSIONS: The results showed that single-staged combined decompression could provide comparable clinical outcomes, and patients' satisfaction was significantly related with postoperative neurological function. In addition, satisfaction score could be decreased by perioperative complications and progression of tandem ossification. Thus, this aggressive surgical strategy should be used more carefully with emphasis on preoperative communication with patients.

[Radiographic comparison of anterior cervical fusion after two-level discectomy or single-level corpectomy for two-level cervical spondylotic myelopathy].

OBJECTIVE: To compare the pre- and post-operative radiographic data of a single-level anterior cervical corpectomy and fusion (ACCF)or a two-level anterior cervical discectomy and fusion (ACDF) for patients with two-level cervical spondylotic myelopathy. METHODS: We retrospectively reviewed the lateral cervical radiographs of 110 patients undergoing a single-level ACCF or a two-level ACDF for the treatment of cervical myelopathy from March 2005 to May 2008. All of them underwent anterior cervical fusion using poly ether ether ketone (PEEK) cage or titanium meshes packed with autogenous bone and fixed-screw titanium plate fixation. A single-level ACCF (group of ACCF, n = 48) or a two-level ACDF (group of ACDF, n = 62) was performed. The following parameters were analyzed: cervical sagittal alignment, Cobb angles of fusion segments, graft collapse, adjacent-segmental degeneration and rate of bone fusion. RESULTS: During a follow-up period of 24 - 60 months, no significant differences existed in sagittal alignment, adjacent-segmental degeneration and rate of bone fusion between two groups. Graft subsidence and loss of Cobb angles of fusion occurred significantly more during the first 2 months post-operation than after 2 months in each group (P < 0.01). However, the group of ACCF subsided and lost more than the group of ACDF (P < 0.05). Caudal endplate subsidence significantly progressed after the first 2 months in the Group of ACCF (P < 0.05). CONCLUSION: Graft subsidence and loss of fusion segmental lordosis of two groups occur mainly in an early post-operation stage (first 2 months). The group of ACDF with PEEK cage is superior to the group of ACCF with titanium meshes in maintaining the height and lordosis of fusion segments. Single-level ACCF with titanium meshes continues subsiding at the caudal endplate of fusion segments even after 2 months.

[Morphologic and apoptotic changes of cervical posterior longitudinal ligament fibroblasts in response to in vitro mechanical stretch].

OBJECTIVE: To investigate the effect of mechanical stretch force on the morphologic and apoptotic changes of fibroblasts derived from the OPLL (ossification of posterior longitudinal ligaments) patients. METHODS: The third passage cells were collected and subjected to 10% elongations cyclic mechanical stretching for 6 h and 24 h with Flexercell 4000(TM) strain unit. Their morphologic changes were observed and the post-stretching apoptotic rates quantified by flow cytometer. The gene expressions of alkaline phosphatase (ALP), collagen types I (COL I) and osteocalcin (OC) were examined. RESULTS: The treated cells were arranged along the vertical direction of force. Stretch force led to a slight increase of apoptosis rate at 6 h and a significant increase of apoptosis rate at 24 h. No significant difference in cellular senescence was observed between control group and treated group. The mRNA expressions of ALP, COL I and OC were positively up-regulated by cyclic stretch at 24 h. CONCLUSION: Stretching force can affect the cellular morphology, promote the osteogenic differentiation and enhance the cellular apoptosis.

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that mechanical strain can lead to the development of OPLL, although the signaling mechanism is not fully understood. Connexin43 (Cx43), a gap-junction protein, has been shown to be of particular importance in bone formation. We hypothesized that Cx43 may play an important role in the signal transmission induced by mechanical strain during the development of OPLL. To explore this possibility, we cultured fibroblasts from spinal ligaments of OPLL and non-OPLL patients and preloaded mechanical stretch onto the cells via a Flexercell 4000 Tension Plus system. We evaluated expression changes in osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 via semi-quantitative RT-PCR and western blotting at 12 and 24 h after mechanical strain application in contrast to static conditions. We observed a significant gene up-regulation of OCN, ALP and COL I and Cx43 protein in OPLL cells after mechanical strain application, but no changes in non-OPLL cells. Notably, after RNA interference targeting Cx43 was performed in OPLL cells, we found that there were no significant changes in the expressions of OCN, ALP, COL I and Cx43 after the mechanical strain was applied for 24 h. Thus, we propose that the increase in Cx43 expression induced by mechanical strain in OPLL cells plays an important role in the progression of OPLL.

Upregulated expression of connexin43 in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

STUDY DESIGN: A case-control study was conducted. OBJECTIVE: To investigate different expressions of connexin43 (Cx43) between spinal ligament fibroblasts from patients with ossification of the posterior longitudinal ligament (OPLL) and non-OPLL patients and demonstrate knockdown of Cx43 protein expression by RNA interference inhibiting expression of osteoblast-specific genes in OPLL cells. SUMMARY OF BACKGROUND DATA: The OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant cellular signaling pathways remain unclear. METHODS: Twenty patients presenting with OPLL and 18 non-OPLL patients underwent anterior decompression between January 2008 and June 2009. Specimens of the posterior longitudinal ligament were collected intraoperatively. Tissue fragment cell culture was performed. Inverted phase contrast microscopy and hematoxylin-eosin staining were used to observe cell morphology. The mouse antivimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence. The messenger RNA expression of osteoblast-specific genes of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of Cx43 was detected via Western blotting. And then, after 72 hours, when RNA interference against Cx43 was performed in OPLL cells, expression of the indexes mentioned earlier was compared again between the transfection group and the nontransfection group. RESULTS: Cultivated cells were observed 7 to 10 days after cell culture. Hematoxylin-eosin staining showed fusiform and multiangular star morphologies, large and elliptical cell nuclei, and ill-defined cell appearances. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The messenger RNA expressions of OCN, ALP, and COL I and protein expressions of Cx43 from OPLL fibroblasts were greater than those from non-OPLL cells, and the difference was significant. Furthermore, knockdown of Cx43 protein expression inhibited the messenger RNA expressions of OCN, ALP, and COL I remarkably in the transfection group compared with the nontransfection group, 72 hours after RNA interference targeting Cx43 was performed in OPLL cells. CONCLUSION: Tissue fragment culture of the cervical posterior longitudinal ligament provided a successful fibroblast culture, showing good adherence and subculture. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, in which Cx43 played an important role.