Objective:To investigate the influence of Kruppel-like factor 16 (KLF16) on the proliferation and migration of pancreatic cancer cells.Methods:Immunohistochemical images of KLF16 were collected from 171 pancreatic cancer tissues and their matched paracarcinoma normal pancreas tissues and 8 pancreatic cancer tissues only in GEPIA database. The expression of KLF16 protein was detected by immunohistochemical imaging software. The protein and mRNA expressions of pancreatic cancer cell lines AsPC-1 and MIA PaCa-2 KLF16 were detected by Western blot and quantitative fluorescence PCR. By knockdown or exogenous overexpression of KLF16, the two cells were divided into blank control group (NC group), negative control group (siRNA-NC group), downexpression KLF16 group (siKLF16 group), overexpression control group (OE-NC group) and ovexpression KLF16-OE group (KLF16-OE group). CCK-8 assay, colony formation assay and transwell chamber were used to detect cell proliferation and migration.Results:The KLF16 protein expression level (4.02±1.26 vs 1.73±1.07) and positive expression rate (91.6% vs 13.5%) in pancreatic cancer tissues were significantly higher than those in paracancer normal pancreas tissues, with statistical significance ( P<0.05). After downregulating KLF16 expression and culturing for 24, 48, 72, and 96 hours, the A450 values of both AsPC-1 (0.19±0.02 vs 0.23±0.03, 0.24±0.06 vs 0.36±0.06, 0.45±0.09 vs 0.78±0.10, 0.69±0.04 vs 0.88±0.07) and MIA PaCa-2 cells (0.20±0.03 vs 0.22±0.02, 0.29±0.05 vs 0.31±0.04, 0.47±0.06 vs 0.78±0.10, 0.71±0.02 vs 0.90±0.07) and colony counts [(36±4.32) per well vs (118.51±10.01) per well, (13.6±2.62) per well vs (83.1±9.11) per well], and the number of migrated cells [(16.67±2.05) vs (46.67±5.91), (19.67±1.69) vs (55±4.89)] all decreased significantly. However, after up-regulating the expression of KLF16 and culturing for 24, 48, 72 and 96 h, the A450 value of both AsPC-1 (0.21±0.05 vs 0.20±0.04, 0.48±0.03 vs 0.31±0.04, 0.91±0.09 vs 0.72±0.03, 1.28±0.10 vs 1.05±0.02) and MIA PaCa-2 cells (0.20±0.01 vs 0.19±0.05, 0.44±0.03 vs 0.30±0.04, 0.89±0.06 vs 0.72±0.03, 1.19±0.05 vs 1.01±0.10), and the number of cell colonies [(189±6.37)/per hole vs (108±9.62)/ per hole, (141±12.56)/ per hole vs (80.69±10.32)/ per hole]], migration cell numbers [(79±4.89) per hole vs (50.33±4.11) per hole, (79.66±3.85) per hole vs (51±4.08) per hole] all increased significantly. Conclusions:KLF16 is highly expressed in pancreatic cancer. The up-regulated expression of KLF16 in pancreatic cancer cell lines can promote the proliferation and migration of pancreatic cancer cells.

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems ; DNA ; Gene Editing/methods* ; Humans ; Streptococcus pyogenes/metabolism*

Objective:To investigate the correlation between paraspinal muscle atrophy, morphological changes of facet joints and adjacent segment disease (ASDis) after lumbar fusion operation.Methods:A retrospective study was conducted among 195 patients who underwent posterior lumbar fusion again for ASDis at this institution from January 2014 to December 2020, including 29 patients with ASDis whose initial surgical fusion segment was L 4,5. According to Roussouly's staging, there were 5 cases of type I, 9 cases of type II, 10 cases of type III, and 5 cases of type IV. Another 29 cases were selected from patients without ASDis after lumbar fusion as a control group. The control group was paired 1∶1 with the ASDis group according to gender, fusion segment, and Roussouly typing of the lumbar spine. The cross-sectional area (CSA) and fat infiltration (FI) of paravertebral muscle, facet joint angle (F-J) and pedicle facet (P-F) angle before the first (second) operation were measured and compared between the two groups. Then logistic regression analysis was used to determine the predictors of ASDis after posterior lumbar fusion. Finally, the receiver operation characteristic (ROC) curve was described, and the area under the curve (AUC) and cut-off point were calculated. At the same time, the paraspinal muscle atrophy before the second operation in ASDis group was measured. Results:The average follow-up time of 98 patients was 59.25±6.38 months (range, 49-73 months). The average body mass index (BMI) of ASDis group was 24.76±3.64 kg/m 2, which was higher than that in control group (22.24±2.92 kg/m 2) ( t=2.481, P=0.041). The average CSA and relative cross-sectional area (rCSA) of paraspinal muscle in ASDis group were 3 214.32± 421.15 mm 2 and 1.69±0.36 respectively, which were less than 3 978.91±459.87 mm 2 and 2.26±0.29 in control group ( t=10.22, P=0.012; t=9.47, P=0.038). The FI degree of paraspinal muscle in ASDis group (21.95%±5.89%) was significantly higher than that in control group (14.64%±7.11%) ( t=7.32, P=0.002). The F-J angle in ASDis group was 35.06°±3.45°, which was less than 38.39°±4.67° in control group ( t=4.76, P=0.027). The P-F angle in ASDis group was 117.39°±8.13°, which was greater than 111.32°±4.78° in control group ( t=5.25, P=0.031). Multivariate logistic regression analysis showed that higher BMI ( OR=1.34, P=0.038), smaller rCSA of paraspinal muscle ( OR=0.02, P=0.017) and higher FI of paraspinal muscle ( OR=1.58, P=0.032) were the risk factors of postoperative ASDis. The ROC curve showed that the AUC of BMI was 0.680 and the cut-off point was 22.58 kg/m 2; The AUC of the FI of paraspinal muscle was 0.716 and the cut-off point was 15.69%; The AUC of rCSA of paraspinal muscle was 0.227 and the cut-off point was 1.92. For ASDis patients, the paraspinal muscle before the second operation had a higher degree of FI (25.47%±6.59% vs. 21.95%±5.89%, t=3.99, P=0.042) and a smaller rCSA (1.52±0.28 vs. 1.69±0.36, t=3.85, P=0.038) than that before the first operation. The difference between the FI degree of paraspinal muscle before the second operation and the first operation was negatively correlated with the occurrence time of ASDis ( r=-0.53, P=0.039) , and the difference of rCSA was positively correlated with the occurrence time of ASDis ( r=0.64, P=0.043) . Conclusion:When BMI >22.58 kg/m 2, FI of paraspinal muscle >15.69%, and rCSA of paraspinal muscle <1.92, it suggests that ASDis is more likely to occur after operation. And the more obvious paraspinal muscle atrophy after the first operation, the earlier ASDis may occur. Morphological changes of facet joints cannot be used as an index to predict the occurrence of ASDis.

Objective:To investigate the influence of interbody cage height during oblique lumbar interbody fusion (OLIF) on lumbar biomechanics with different degrees of degeneration and to provide a reference for cage choice.Methods:The finite element model of normal lower lumbar spine (L 3-S 1) was built and validated, then constructed three different degenerative segments in L 3, 4, and the cages with different height (8, 10,12, 14 mm) were implanted into L 4, 5 disc. All the twelve models were loaded with pure moment of 7.5 N·m to produce flexion, extension, lateral bending and axial rotation motions on lumbar spine, and the effects of cage height on range of motion (ROM), intervertebral pressure in adjacent segments and stress in facet joints were investigated. Results:The ROM of adjacent segments and the maximum stress of intervertebral discs increased with the increase of cage height, but this trend was not obvious in moderate and severe degeneration groups. After implantation of 4 different height cages (8, 10, 12, 14 mm), the ROM of L 3, 4 segment reached the maximum during extension. The ROM of mild degeneration group was 2.68 °, 2.71 °, 2.94 °, 2.98 °, moderate degeneration group was 2.33°, 2.37°, 2.41°, 2.49°, and severe degeneration group was 1.94 °, 1.99 °, 2.14 °, 2.21 °. The stress of L 3, 4 intervertebral disc reached the maximum during right bending. The maximum stress of L 3, 4 intervertebral disc was 23.95 MPa, 24.60 MPa, 24.90 MPa and 25.34 MPa in mild group, 25.57 MPa, 25.60 MPa, 25.82 MPa and 25.89 MPa in moderate group, and 25.95 MPa, 25.99 MPa, 26.48 MPa and 27.13 MPa in severe group. The maximum stress of L 3, 4 facet joint was 15.87 MPa, 15.78 MPa, 16.29 MPa and 16.43 MPa in mild group, 15.97 MPa, 16.31 MPa, 16.53 MPa and 16.79 MPa in moderate group, and 16.17 MPa, 16.49 MPa, 16.95 MPa and 17.35 MPa in severe group. Conclusion:For patients with mild lumbar degeneration requiring OLIF surgery, the intervertebral height of the surgical segment should not be overstretched. But for patients with moderate to severe lumbar degenerative disease who need to undergo OLIF surgery, it is recommended that the cage height be 0-2 mm higher than the original intervertebral space height.

Objective:To explore the effect of intravascular sodium nitroprusside (SNP) combined with hyaluronidase (HAase) plus urokinase (UK) in treating rat abdominal wall skin flap ischemia caused by HA induced artery embolism, and to compare the difference between thrombolysis with and without addition of sodium nitroprusside.Methods:Forty male Sprague-Dawley rats were injected with 10 μl of hyaluronic acid (HA) into the left inferior epigastric arteries, constructing the superficial epigastricl artery skin flap ischemic model in rats. The rats were randomly divided into four groups: a control group, and experimental groups A, B and C. Control and experimental groups A, B and C were treated with the following solutions 45 minutes after hyaluronic acid injection: physiological saline plus glucose was injected into the rats (control group); hyaluronidase (HAase) plus glucose injection was injected into the rats (experimental group A), hyaluronidase (HAase) plus urokinase (UK) was injected into the rats (experimental group B), hyaluronidase (HAase), urokinase (UK) plus sodium nitroprusside were injected into the rats (experimental group C). The changes of flaps were observed at 0 min, 3 days, 5 days and 7 days after operation. The difference of the area percentage of unperfused flap in the four groups was compared 7 days after operation. This study was carried out from July 2020 to March 2021 in the Medical Laboratory Animal Center of Weifang Medical University.Results:The unperfused area of flaps for the control group, experimental groups A, B and C were (100.00±0.00) %, (44.68±7.90)%, (34.01±8.77)% and (24.12±4.58)%, respectively. In the experimental group C, the scabby necrosis area was smaller than that of the experimental group A ( P<0.05); in the experimental group C, the scabby necrosis area was smaller than that of the experimental group B ( P<0.05); in the experimental group B, the scabby necrosis area was smaller than that of the experimental group A ( P<0.05). HE staining revealed that size and density of the embolus was significantly decreased after the addition of sodium nitroprusside. Conclusions:Sodium nitroprusside combined with hyaluronidase and urokinase can effectively improve the ischemia of the flap caused by HA induced artery embolism, increase the tissue perfusion, and reduce the necrotic area of the flap.

Objective:To investigate the efficacy of continuous intravenous injection of hyaluronidase (HAase) combined with urokinase (UK) in the treatment of ischemia of hyaluronic acid (HA) arterial embolized skin flap.Methods:(1)Establish an animal model of superficial abdominal artery embolization with HA: 24 SD rats were used to make a square island flap (2 cm×2 cm, the width of the pedicle is 2 mm) on the left and right sides of the abdominal white line with superficial epigastric artery as feeding artery. The left and right flaps of rats were used as experimental group and control group. After the preparation of the experimental skin flap, HA 10 μl was injected into the superficial abdominal artery, while the self-control flap was only made and the vessels were peeled off without embolization.(2)The rats were randomly divided into A, B, C and D groups by drawing lots with 6 rats in each group, After the successful establishment of embolic animal model of 45 min, HAase(2 000 IU/kg)+ UK(50 000 IU/kg), HAase(2 000 IU/kg), UK(50 000 IU/kg) and normal saline were continuously infused through caudal vein with microinjection pump. The volume of solution in each group was 6 ml in 12 minutes. The general condition of rats and the skin color, edema, congestion and skin necrosis in the operation area of rats were observed, which were photographed and compared immediately, and at 3 days, 5 days and 7 days after embolization. The images were analyzed by Photoshop software, and the percentage of survival area of the flap after operation 7 days, was measured by pixel method. The percentage of survival area was compared by single factor analysis of variance (ANOVA) and head-to-head comparison by LSD- t test. Results:Immediately after operation, all the flaps in the four groups were pale and there was no obvious swelling. All the flaps showed different degrees of swelling within 3 days after operation, and the swelling basically disappeared within 3-5 days after operation, and gradually changed from dark red ecchymosis to dark purple or black. Seven days after operation, the necrotic area flap gradually hardened and its boundary was obvious. The skin flaps of the 4 groups showed different degrees of necrosis. The postoperative reaction of the flap was the slightest in group A, the performance of group B and C was similar, which were both between group A and group D, and there was the heaviest postoperative reaction in group D. At 7 days after operation, the percentage of survival area of flaps in groups A, B, C and D was 90.30%±5.95%, 52.63%±6.90%, 51.14%±5.95% and 7.70%±2.18%, respectively. The percentage of survival area of skin flap in group A was significantly higher than that in groups B, C and D (analysis of ANOVA: P<0.01; LSD- t test: P<0.01). Conclusions:Continuous intravenous infusion of HAase combined with UK can effectively alleviate the flap ischemia caused by HA artery embolism, increase tissue perfusion and increase the survival area of the flap.

Objective:To investigate the efficacy of continuous intravenous injection of hyaluronidase (HAase) combined with urokinase (UK) in the treatment of ischemia of hyaluronic acid (HA) arterial embolized skin flap.Methods:(1)Establish an animal model of superficial abdominal artery embolization with HA: 24 SD rats were used to make a square island flap (2 cm×2 cm, the width of the pedicle is 2 mm) on the left and right sides of the abdominal white line with superficial epigastric artery as feeding artery. The left and right flaps of rats were used as experimental group and control group. After the preparation of the experimental skin flap, HA 10 μl was injected into the superficial abdominal artery, while the self-control flap was only made and the vessels were peeled off without embolization.(2)The rats were randomly divided into A, B, C and D groups by drawing lots with 6 rats in each group, After the successful establishment of embolic animal model of 45 min, HAase(2 000 IU/kg)+ UK(50 000 IU/kg), HAase(2 000 IU/kg), UK(50 000 IU/kg) and normal saline were continuously infused through caudal vein with microinjection pump. The volume of solution in each group was 6 ml in 12 minutes. The general condition of rats and the skin color, edema, congestion and skin necrosis in the operation area of rats were observed, which were photographed and compared immediately, and at 3 days, 5 days and 7 days after embolization. The images were analyzed by Photoshop software, and the percentage of survival area of the flap after operation 7 days, was measured by pixel method. The percentage of survival area was compared by single factor analysis of variance (ANOVA) and head-to-head comparison by LSD- t test. Results:Immediately after operation, all the flaps in the four groups were pale and there was no obvious swelling. All the flaps showed different degrees of swelling within 3 days after operation, and the swelling basically disappeared within 3-5 days after operation, and gradually changed from dark red ecchymosis to dark purple or black. Seven days after operation, the necrotic area flap gradually hardened and its boundary was obvious. The skin flaps of the 4 groups showed different degrees of necrosis. The postoperative reaction of the flap was the slightest in group A, the performance of group B and C was similar, which were both between group A and group D, and there was the heaviest postoperative reaction in group D. At 7 days after operation, the percentage of survival area of flaps in groups A, B, C and D was 90.30%±5.95%, 52.63%±6.90%, 51.14%±5.95% and 7.70%±2.18%, respectively. The percentage of survival area of skin flap in group A was significantly higher than that in groups B, C and D (analysis of ANOVA: P<0.01; LSD- t test: P<0.01). Conclusions:Continuous intravenous infusion of HAase combined with UK can effectively alleviate the flap ischemia caused by HA artery embolism, increase tissue perfusion and increase the survival area of the flap.

Biliary stent has been widely used in the treatment of biliary stricture and obstruction, it can relieve the pain of patients effectively, but bacterial infection and stent obstruction are still troublesome after surgery. We introduce the mechanism of infection and stent blockage caused by bacterial invasion after biliary stent implantation, and expound the formation mechanism of bacterial biofilm and bile sludge in this review. Antibacterial biliary stent is an effective way to inhibit biliary tract infection, the literatures on antibacterial modification of biliary stent with different antibacterial methods in domestic and abroad are reviewed, and the research prospect of antibacterial biliary stent is summarized and prospected.
Anti-Bacterial Agents/pharmacology* ; Bile ; Biliary Tract ; Cholestasis ; Humans ; Stents

Objective:To compare the effects of the fracture line direction in the sagittal profile on the Gartland type Ⅲ supracondylar humerus fractures in children.Methods:A total of 153 cases of Gartland type Ⅲ supracondylar humerus fractures treated in the Department of Pediatric Orthopedics, Qilu Hospital of Shandong University (Qingdao) from January 2017 to April 2019 were retrospectively analyzed.They were categorized into the traditional oblique type (the fracture line went downward in the front and upward in the back), transverse type (the fracture line went horizontally) and reverse oblique type (the fracture line went upward in the front and downward in the back). Gender, age, injury side, cause of injury, ecchymosis before elbow, preoperative neurological symptoms, operation time, incision ratio, and Flynn scores of the elbow joint at the last follow-up were compared among the 3 groups.Results:Patients were followed up for (8.65±2.47) months (6-15 months). There were 60, 64 and 29 patients in the traditional oblique type, transverse type and reserve oblique type groups, respectively.There were no differences in the gender, injury side, and injury causes among the 3 groups (all P>0.05). The age of the traditional oblique type, transverse type and reverse oblique type group were (4.76±2.51) years, (4.71±2.09) years and (6.32±1.98) years, respectively, which was significant different among the 3 groups ( F=5.826, P<0.05). There were 10, 7 and 11 cases of preoperative elbow ecchymosis occurred in children of the traditional oblique type, transverse type and reverse oblique type groups, respectively, which was significant different ( χ2=9.902, P<0.05). No significant differences were found in preoperative neurological symptoms of the 3 groups ( P>0.05). The operative time for the traditional oblique type, transverse type and reverse oblique type group were (43.28±24.25) min, (40.95±27.41) min and (58.66±34.08) min, which was significant different ( F=4.337, P<0.05). The traditional oblique type and transverse type groups had 1 failure case of closed reduction, respectively, and the incision was performed during the operation.There were 4 cases in the reverse oblique type group who underwent the open reduction.The reduction rate was significantly different among 3 groups ( χ2=6.883, P<0.05). There was no significant difference in the excellent to good rate of traditional oblique type (96.67%, 58/60 cases), transverse type(95.31%, 61/64 cases)and reserve oblique type (93.10%, 27/29 cases) among 3 groups ( P>0.05). Conclusions:The reverse oblique Gartland type Ⅲ supracondylar humerus fractures are relatively rare in clinical practice, which involves more severe soft tissue damages and more obvious antecubital ecchymosis.The conventional reduction methods seem to be ineffectual for the reverse oblique supracondylar humerus fractures.

Objective:To explore the relationship among the supratrochlear and supraorbital neurovascular bundles and the corrugator muscle, in order to provide anatomical basis for eyebrow operations.Methods:From July to October 2019, the upper eyelids and forehead of 10 adult skull specimens (20 sides) were dissected layer by layer in the anatomy laboratory of Weifang Medical College. The line of bilateral inner canthus was taken as the X-axis and the facial midline as the Y-axis to establish the coordinate system. The coordinate parameters of supratrochlear nerve, supratrochlear artery, medial branch of supraorbital nerve, lateral branch of supraorbital nerve and supraorbital artery were measured to obtain the range of its trajectory, and their level and their adjacent relationship with the corrugator muscle were observed.Results:The corrugator muscle started from the upper part of the nasal process of the frontal bone near the medial margin of the orbit and was (4.32±0.98) mm from the midline. The base of the corrugator muscle start was (9.91±1.41) mm wide. The corrugator muscle run obliquely upward, through orbicularis oculi muscle and frontal muscle, and stopped at subcutaneous of the outer 1/3 of eyebrow. The trapezoid area was composed of the line of 15 mm above the inner canthus line, (16.58±1.70) mm and (16.17±1.42) mm from the facial midline as the lower base and the line of 30 mm above the inner canthus line, (13.74±1.54) mm and (14.21±2.42) mm from the facial midline, which was the dangerous area for the supratrochlear neurovascular bundles walking in corrugator muscle. The trapezoid area was composed of the line of 22.5 mm above the inner canthus line, (26.03±1.55) mm and (28.11±2.46) mm from the facial midline as the lower base and the line of 40 mm above the inner canthus line, (31.23±3.19) mm and (38.20±2.94) mm from the facial midline, which was the dangerous area for the supraorbital neurovascular bundles.Conclusions:Familiar with the relationship among the supratrochlear and supraorbital neurovascular bundle and the corrugator muscle can avoid dangerous areas and reduce superficial and deep neurovascular injury during operation.