Human keratinocyte growth factor-1 (hKGF-1), a member of the fibroblast growth factor (FGF) family, plays crucial roles in organ development, cell proliferation, wound healing, and tissue repair, representing one of the most effective and specific growth factors for skin repair. However, obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro. This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1 (hKGF-1) protein, ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line (BmN). In this study, we optimized the hKGF-1 sequence based on the codon preference of baculovirus. By fusing hKGF-1 with polyhedrin (highly expressed in this system) and adding extra promoters and enhancers, we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells. The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells. SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression (accounting for 8.7% of total cellular protein) was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression. The optimal harvest time was determined to be 120 h post transfection. This study achieved the efficient expression of hKGF-1 in Bombyx mori cells, establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1. The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins, but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
Bombyx/metabolism* ; Animals ; Baculoviridae/metabolism* ; Humans ; Fibroblast Growth Factor 7/biosynthesis* ; Recombinant Proteins/genetics* ; Cell Line ; Genetic Vectors/genetics*

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.
Humans ; Epidermal Growth Factor/biosynthesis* ; Genetic Vectors/genetics* ; Recombinant Fusion Proteins/biosynthesis* ; Animals ; Bombyx/metabolism* ; Occlusion Body Matrix Proteins/genetics* ; Nucleopolyhedroviruses/genetics* ; Amino Acid Sequence

Objective:To explore the clinicopathological and prognostic features of young onset patients with middle-low rectal cancer who received neoadjuvant chemoradiotherapy (NCRT).Methods:After NCRT, a total of 441 patients with primary middle-low rectal cancer treated with radical surgery at the Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS) from January 2004 to December 2016 were included. According to the age of disease onset, the patients were divided into the young group (51cases) and the middle-old group (390 cases), and the clinicopathological characteristics and survival of these patients were analyzed.Results:In the young group, 68.6% of patients received radical surgery within 7 weeks after NCRT, which was higher than 52.8% in the middle-old group ( P=0.047). The stage ypTNM Ⅲ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.027). The stage ypN+ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.047), The incidence of disease progression in the young group was 39.2%, higher than 25.1% in the middle-old group ( P=0.049). The incidence of distant metastasis in the young group was 35.3%, higher than 21.5% in the middle-old group( P=0.044). Most cases of disease progression occurred in the first 3 years after surgery for the young group, especially in the second year after surgery, the incidence of disease progression in the young group was 55.0%, higher than 26.5% in middle-old group ( P=0.025). The 3-year and 5-year disease-free survival (DFS) rates for the young group were 63.7% and 58.2%, lower than 81.0% and 74.3% in the middle-old group ( P=0.016), respectively. The 3-year and 5-year overall survival in the middle-old group (OS) rates for the young group were 85.4% and 69.2%, lower than 93.6% and 84.1% in the middle-old group ( P=0.033), respectively. The multivariate analysis showed that, response of primary tumor ( HR=4.804, 95% CI: 1.360-16.973) and total number of dissected lymph nodes ( HR=4.336, 95% CI: 1.739-10.809) in the young group were independent prognostic factors related to DFS. The total dissected number of lymph nodes( HR=3.295, 95% CI: 1.076-10.091)was an independent prognostic factor related to OS. In the middle-old group, response of primary tumor ( HR=2.626, 95% CI: 1.354-5.091), ypTNM stage (ypTNM Ⅲ: HR=5.837, 95% CI: 2.968-11.479) and tumor location distance from the anal verge ( HR=0.500, 95% CI: 0.308-0.812) were independent prognostic factors related to DFS. Lymphovascular invasion ( HR=0.500, 95% CI: 0.308-0.812) and ypTNM stage (ypTNM Ⅲ: HR=16.322, 95% CI: 5.049-52.771) were independent prognostic factors related to OS. Conclusions:Young onset rectal cancer patients are associated with shorter operation time interval, advanced pathological stage and poorer prognosis. More intensive adjuvant treatment and post-treatment surveillance should be conducted to young onset rectal cancer with NCRT.

Objective:To explore the risk factors for regional lymph node (RLN) metastasis in colorectal cancer patients with mismatch repair deficiency (dMMR).Methods:The data of 357 dMMR colorectal cancer patients who underwent surgery in National Cancer Center from January 2012 to December 2016 was retrospectively analyzed. Univariate and multivariate analysis were used to identify the risk factors for RLN metastasis.Results:Among the 357 patients, 204 were male and 153 were female, 61.6% (220/357) lesion located in right half colon, while the other 16.2% (58/357) located in rectum. Univariate analysis showed that tumor size, differentiation, lymphovascular invasion, tumor deposit, postoperative pathologic T stage (pT), the number of negative lymph nodes and the expression of the MSH6 protein were significantly associated with RLN metastasis ( P<0.05). All of the patients with well differentiation tumors (15 patients) or staged pT1 (13 patients) had no RLN metastasis. Multivariate analysis showed that tumor differentiation ( OR=2.582, 95% CI=1.567-4.274, P<0.001), pT ( OR=3.778, 95% CI=1.448-12.960, P=0.015) and the expression of MSH6 protein ( OR=2.188, 95% CI=1.159-4.401, P=0.021) were independent risk factors for RLN metastasis. Conclusions:The postoperative pT stage, tumor differentiation and the expression of MSH6 protein are independent risk factors for RLN metastasis of dMMR colorectal cancer. Preoperative assessment of these factors may further improve the accuracy of predicting the risk of RLN metastasis.

Objective:To explore the application value of the conditional disease-free survival (cDFS) analysis in predicting prognosis of stage-specific rectal cancer patients underwent neoadjuvant chemoradiotherapy (nCRT).Methods:Clinicopathologic data of 436 patients with rectal cancer received nCRT and radical operation in Cancer Hospital, Chinese Academy of Medical Sciences between January 2004 and December 2016 were retrospectively reviewed. With reference to conditional probability, the 3-year cDFS of patients at different ypTNM stage after completion of nCRT was estimated using the Kaplan-Meier method.Results:There were 66 patients of ypTNM stage 0 (pathological complete response), 87 patients of ypTNM stage Ⅰ, 135 patients of ypTNM stage Ⅱ and 148 patients of ypTNM stage Ⅲ. The 3-year accumulated DFS of patients with ypTNM stage 0, ypTNM stage Ⅰ, ypTNM stage Ⅱ, and ypTNM stage Ⅲ were 97.0%, 93.1%, 85.2%, and 64.2%, respectively. On the condition of postoperactive disease-free survival for 1 year, 2 years, 3 years, 4 years, and 5 years, the corresponding 3-year cDFS of patients at ypTNM stage 0 were 97.0%, 95.5%, 96.9%, 98.4%, 100.0%, respectively. The corresponding 3-year cDFS of patients at ypTNM Ⅲ were 68.2%, 79.3%, 86.3%, 92.1%, 96.4%, respectively. The more advanced ypTNM staging resulted in the more improvement of 3-year cDFS being acquired.Conclusion:cDFS is a better method to reflect the dynamic changes of the prognosis of rectal cancer patients with nCRT in different ypTNM stage, and it is useful to guide the clinicians to assess the prognosis and propose appropriate surveillance.

Objective:To explore the clinicopathological and prognostic features of young onset patients with middle-low rectal cancer who received neoadjuvant chemoradiotherapy (NCRT).Methods:After NCRT, a total of 441 patients with primary middle-low rectal cancer treated with radical surgery at the Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS) from January 2004 to December 2016 were included. According to the age of disease onset, the patients were divided into the young group (51cases) and the middle-old group (390 cases), and the clinicopathological characteristics and survival of these patients were analyzed.Results:In the young group, 68.6% of patients received radical surgery within 7 weeks after NCRT, which was higher than 52.8% in the middle-old group ( P=0.047). The stage ypTNM Ⅲ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.027). The stage ypN+ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.047), The incidence of disease progression in the young group was 39.2%, higher than 25.1% in the middle-old group ( P=0.049). The incidence of distant metastasis in the young group was 35.3%, higher than 21.5% in the middle-old group( P=0.044). Most cases of disease progression occurred in the first 3 years after surgery for the young group, especially in the second year after surgery, the incidence of disease progression in the young group was 55.0%, higher than 26.5% in middle-old group ( P=0.025). The 3-year and 5-year disease-free survival (DFS) rates for the young group were 63.7% and 58.2%, lower than 81.0% and 74.3% in the middle-old group ( P=0.016), respectively. The 3-year and 5-year overall survival in the middle-old group (OS) rates for the young group were 85.4% and 69.2%, lower than 93.6% and 84.1% in the middle-old group ( P=0.033), respectively. The multivariate analysis showed that, response of primary tumor ( HR=4.804, 95% CI: 1.360-16.973) and total number of dissected lymph nodes ( HR=4.336, 95% CI: 1.739-10.809) in the young group were independent prognostic factors related to DFS. The total dissected number of lymph nodes( HR=3.295, 95% CI: 1.076-10.091)was an independent prognostic factor related to OS. In the middle-old group, response of primary tumor ( HR=2.626, 95% CI: 1.354-5.091), ypTNM stage (ypTNM Ⅲ: HR=5.837, 95% CI: 2.968-11.479) and tumor location distance from the anal verge ( HR=0.500, 95% CI: 0.308-0.812) were independent prognostic factors related to DFS. Lymphovascular invasion ( HR=0.500, 95% CI: 0.308-0.812) and ypTNM stage (ypTNM Ⅲ: HR=16.322, 95% CI: 5.049-52.771) were independent prognostic factors related to OS. Conclusions:Young onset rectal cancer patients are associated with shorter operation time interval, advanced pathological stage and poorer prognosis. More intensive adjuvant treatment and post-treatment surveillance should be conducted to young onset rectal cancer with NCRT.

Objective:To explore the risk factors for regional lymph node (RLN) metastasis in colorectal cancer patients with mismatch repair deficiency (dMMR).Methods:The data of 357 dMMR colorectal cancer patients who underwent surgery in National Cancer Center from January 2012 to December 2016 was retrospectively analyzed. Univariate and multivariate analysis were used to identify the risk factors for RLN metastasis.Results:Among the 357 patients, 204 were male and 153 were female, 61.6% (220/357) lesion located in right half colon, while the other 16.2% (58/357) located in rectum. Univariate analysis showed that tumor size, differentiation, lymphovascular invasion, tumor deposit, postoperative pathologic T stage (pT), the number of negative lymph nodes and the expression of the MSH6 protein were significantly associated with RLN metastasis ( P<0.05). All of the patients with well differentiation tumors (15 patients) or staged pT1 (13 patients) had no RLN metastasis. Multivariate analysis showed that tumor differentiation ( OR=2.582, 95% CI=1.567-4.274, P<0.001), pT ( OR=3.778, 95% CI=1.448-12.960, P=0.015) and the expression of MSH6 protein ( OR=2.188, 95% CI=1.159-4.401, P=0.021) were independent risk factors for RLN metastasis. Conclusions:The postoperative pT stage, tumor differentiation and the expression of MSH6 protein are independent risk factors for RLN metastasis of dMMR colorectal cancer. Preoperative assessment of these factors may further improve the accuracy of predicting the risk of RLN metastasis.

Objective:To explore the application value of the conditional disease-free survival (cDFS) analysis in predicting prognosis of stage-specific rectal cancer patients underwent neoadjuvant chemoradiotherapy (nCRT).Methods:Clinicopathologic data of 436 patients with rectal cancer received nCRT and radical operation in Cancer Hospital, Chinese Academy of Medical Sciences between January 2004 and December 2016 were retrospectively reviewed. With reference to conditional probability, the 3-year cDFS of patients at different ypTNM stage after completion of nCRT was estimated using the Kaplan-Meier method.Results:There were 66 patients of ypTNM stage 0 (pathological complete response), 87 patients of ypTNM stage Ⅰ, 135 patients of ypTNM stage Ⅱ and 148 patients of ypTNM stage Ⅲ. The 3-year accumulated DFS of patients with ypTNM stage 0, ypTNM stage Ⅰ, ypTNM stage Ⅱ, and ypTNM stage Ⅲ were 97.0%, 93.1%, 85.2%, and 64.2%, respectively. On the condition of postoperactive disease-free survival for 1 year, 2 years, 3 years, 4 years, and 5 years, the corresponding 3-year cDFS of patients at ypTNM stage 0 were 97.0%, 95.5%, 96.9%, 98.4%, 100.0%, respectively. The corresponding 3-year cDFS of patients at ypTNM Ⅲ were 68.2%, 79.3%, 86.3%, 92.1%, 96.4%, respectively. The more advanced ypTNM staging resulted in the more improvement of 3-year cDFS being acquired.Conclusion:cDFS is a better method to reflect the dynamic changes of the prognosis of rectal cancer patients with nCRT in different ypTNM stage, and it is useful to guide the clinicians to assess the prognosis and propose appropriate surveillance.

Objective:To analyze the clinical-pathological data of patients with locally advanced rectal cancer who underwent modified total neoadjuvant therapy (TNT), and to evaluate the safety and efficacy of radical surgery after modified total neoadjuvant therapy.Methods:The clinical-pathological data of 30 locally advanced rectal cancer patients who underwent modified TNT (mTNT) followed by radical resection were retrospectively analyzed. The surgical procedure, postoperative complications, tumor regression grade, tumor downstaging and prognosis were analyzed.Results:The 30 patients included 24 males and 6 females with a median age of 55.5 years. All patients underwent radical surgery after neoadjuvant therapy, 14 patients received low anterior resection, 14 patients received abdominal perineal resection, and the other 2 patients received Hartmann procedure. All patients achieved R0 resection with a median operative time 220 minutes and the median intraoperative blood loss was 200 ml. The morbidity of postoperative complications was 20% (6/30), including dysuria in 2 patients, delayed healing of perineal incision in 2 patients, intestinal obstruction in 1 patient and pelvic hemorrhage in 1 patient. The median time to first flatus after surgery was 3 days and the median postoperative hospital stay was 8 days. Postoperative pathological results showed that 15 patients (50.0%) had severe tumor regression, including 4 patients (13.3%) achieved pathological complete response (pCR), 12 patients (40.0%) had moderate tumor regression, and 3 patients (10.0%) had minor tumor regression. Twenty patients had detailed pre-treatment clinical stage, and among those 20 patients, 15 patients (75.0%) and 13 patients (65.0%) achieved downstaging of tumor T stage and N stage, respectively. Only 2 patients appeared distant metastasis, and no patient had local recurrence.Conclusions:For locally advanced rectal cancer patients, mTNT doesn′t increase the morbidity of postoperative complication and is a safe and effective treatment strategy with satisfactory short-term result.

Objective:To analyze the clinical-pathological data of patients with locally advanced rectal cancer who underwent modified total neoadjuvant therapy (TNT), and to evaluate the safety and efficacy of radical surgery after modified total neoadjuvant therapy.Methods:The clinical-pathological data of 30 locally advanced rectal cancer patients who underwent modified TNT (mTNT) followed by radical resection were retrospectively analyzed. The surgical procedure, postoperative complications, tumor regression grade, tumor downstaging and prognosis were analyzed.Results:The 30 patients included 24 males and 6 females with a median age of 55.5 years. All patients underwent radical surgery after neoadjuvant therapy, 14 patients received low anterior resection, 14 patients received abdominal perineal resection, and the other 2 patients received Hartmann procedure. All patients achieved R0 resection with a median operative time 220 minutes and the median intraoperative blood loss was 200 ml. The morbidity of postoperative complications was 20% (6/30), including dysuria in 2 patients, delayed healing of perineal incision in 2 patients, intestinal obstruction in 1 patient and pelvic hemorrhage in 1 patient. The median time to first flatus after surgery was 3 days and the median postoperative hospital stay was 8 days. Postoperative pathological results showed that 15 patients (50.0%) had severe tumor regression, including 4 patients (13.3%) achieved pathological complete response (pCR), 12 patients (40.0%) had moderate tumor regression, and 3 patients (10.0%) had minor tumor regression. Twenty patients had detailed pre-treatment clinical stage, and among those 20 patients, 15 patients (75.0%) and 13 patients (65.0%) achieved downstaging of tumor T stage and N stage, respectively. Only 2 patients appeared distant metastasis, and no patient had local recurrence.Conclusions:For locally advanced rectal cancer patients, mTNT doesn′t increase the morbidity of postoperative complication and is a safe and effective treatment strategy with satisfactory short-term result.